Using nucleosome interacting protein domains to enhance targeted genome modification

ABSTRACT

Compositions and methods for using nucleosome interacting protein domains to increase accessibility of programmable DNA modification proteins to target chromosomal sequences, thereby increasing efficiency of targeted genome/epigenetic modification in eukaryotic cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/031,819, filed Jul. 10, 2018, which claims priority to U.S.Provisional Application Ser. No. 62/531,222, filed Jul. 11, 2017, thedisclosure of each of which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jul. 6, 2018, is named599979_SequenceListing_ST25.txt, and is 264 kilobytes in size.

FIELD

The present disclosure relates to compositions and methods forincreasing the efficiency of targeted genome modification, targetedtranscriptional regulation, or targeted epigenetic modification.

BACKGROUND

Programmable endonucleases have increasingly become an important toolfor targeted genome engineering or modification in eukaryotes. Recently,RNA-guided clustered regularly interspersed short palindromic repeats(CRISPR) systems have emerged as a new generation of genome modificationtools. These new programmable endonucleases provide unprecedentedsimplicity and versatility as compared to previous generations ofnucleases such as zinc finger nucleases (ZFNs) and transcriptionactivator-like effector nucleases (TALENs). However, chromatin barriersin eukaryotic cells can hinder target access and cleavage by theprokaryote-derived CRISPR systems (Hinz et al., Biochemistry, 2015,54:7063-66; Horlbeck et al., eLife, 2016, 5:e12677).

In fact, no or low editing activity on certain mammalian genomic siteshas been observed when using Streptococcus pyogenes Cas9 (SpCas9), whichis considered the most active CRISPR nuclease to date. Moreover, many ofthe CRISPR nucleases that have been characterized thus far exhibit noactivity in mammalian cells, even though they are active in bacteria oron purified DNA substrates. Therefore, there is a need to improve theability of CRISPR nuclease systems and other programmable DNAmodification proteins to overcome chromatin hindrance to increase theefficiency of targeted genome or epigenetic modification in eukaryotes.

SUMMARY

Among the various aspects of the present disclosure is the provision offusion proteins, wherein each fusion protein comprises a CRISPR proteinlinked to at least one nucleosome interacting protein domain.

In general, the CRISPR protein can be a type II CRISPR/Cas9 protein or atype V CRISPR/Cpf1 protein. In certain embodiments, the CRISPR proteincan be Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilusCas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacterjejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9), Francisellanovicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), Neisseriameningitis Cas9 (NmCas9), Francisella novicida Cpf1 (FnCpf1),Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006Cpf1 (LbCpf1).

In some embodiments, the CRISPR protein has nuclease or nickaseactivity. For example, the CRISPR protein can be a type II CRISPR/Cas9nuclease or nickase, or a type V CRISPR/Cpf1 nuclease or nickase. Inother embodiments, the CRISPR protein has non-nuclease activity. In suchiterations, the CRISPR protein can be a type II CRISPR/Cas9 proteinmodified to lack all nuclease activity and linked to a non-nucleasedomain, or a type V CRISPR/Cpf1 protein modified to lack all nucleaseactivity and linked to a non-nuclease domain, wherein the non-nucleasedomain can have cytosine deaminase activity, histone acetyltransferaseactivity, transcriptional activation activity, or transcriptionalrepressor activity.

The at least one nucleosome interacting protein domain of the fusionprotein can be a high mobility group (HMG) box (HMGB) DNA bindingdomain, a HMG nucleosome-binding (HMGN) protein, a central globulardomain from a histone H1 variant, a DNA binding domain from a chromatinremodeling complex protein, or a combination thereof. In certainembodiments, the at least one nucleosome interacting protein domain ofthe fusion protein can be HMGB1 box A domain, HMGN1 protein, HMGN2protein, HMGN3a protein, HMGN3b protein, histone H1 central globulardomain, imitation switch (ISWI) protein DNA binding domain,chromodomain-helicase-DNA protein 1 (CHD1) DNA binding domain, or acombination thereof.

The at least one nucleosome interacting protein domain can be linked tothe CRISPR protein directly via a chemical bond, indirectly via alinker, or a combination thereof. The at least one nucleosomeinteracting protein domain can be linked to the N-terminus, C-terminus,and/or an internal location of the CRISPR protein. In some embodiments,the fusion protein comprises at least two nucleosome interacting proteindomains linked to the CRISPR protein.

The fusion proteins disclosed herein can further comprise at least onenuclear localization signal, at least one cell-penetrating domain, atleast one marker domain, or a combination thereof.

In certain embodiments, the fusion protein can have an amino acidsequence having at least about 90% sequence identity with SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79.

In other embodiments, the fusion protein can have an amino acid sequenceas set forth in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74,SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79.

Another aspect of the present disclosure encompasses protein-RNAcomplexes comprising at least one of the CRISPR-containing fusionproteins disclosed herein and at least one guide RNA.

A further aspect of the present disclosure provides nucleic acidsencoding any of the fusion proteins disclosed herein. The nucleic acidscan be codon optimized for translation in a eukaryotic cell. In someembodiments, the nucleic acids can be part of a vector such as, forexample, a viral vector, a plasmid vector, or a self-replicating RNA.

Still another aspect of the present disclosure provides methods forincreasing efficiency of targeted genome or epigenetic modification in aeukaryotic cell. The methods involve introducing into a eukaryotic cell(a) at least one fusion protein or nucleic acid encoding at least onefusion protein, each fusion protein comprising a CRISPR protein linkedto at least one nucleosome interacting protein domain, wherein theCRISPR protein (i) has nuclease or nickase activity or (ii) is modifiedto lack all nuclease activity and is linked to a non-nuclease domain;and (b) at least one guide RNA or nucleic acid encoding at least oneguide RNA; wherein the CRISPR protein of the at least one fusion proteinis targeted to a target chromosomal sequence and the at least onenucleosome interacting protein domain of the at least one fusion proteinalters nucleosomal or chromatin structure such that the at least onefusion protein has increased access to the target chromosomal sequence,thereby increasing efficiency of targeted genome or epigeneticmodification.

In general, the CRISPR protein of the fusion protein used in the methodsdisclosed herein can be a type II CRISPR/Cas9 protein or a type VCRISPR/Cpf1 protein. In embodiments in which the CRISPR protein hasnuclease or nickase activity, the CRISPR protein can be a type IICRISPR/Cas9 nuclease or nickase, or a type V CRISPR/Cpf1 nuclease ornickase. In embodiments in which the CRISPR protein has non-nucleaseactivity, the CRISPR protein can be a type II CRISPR/Cas9 proteinmodified to lack all nuclease activity and linked to a non-nucleasedomain, or a type V CRISPR/Cpf1 protein modified to lack all nucleaseactivity and linked to a non-nuclease domain, wherein the non-nucleasedomain can have cytosine deaminase activity, histone acetyltransferaseactivity, transcriptional activation activity, or transcriptionalrepressor activity.

The at least one nucleosome interacting protein domain of the fusionprotein used in the methods can be a high mobility group (HMG) box(HMGB) DNA binding domain, a HMG nucleosome-binding (HMGN) protein, acentral globular domain from a histone H1 variant, a DNA binding domainfrom a chromatin remodeling complex protein, or a combination thereof.In certain embodiments, the at least one nucleosome interacting proteindomain of the fusion protein can be HMGB1 box A domain, HMGN1 protein,HMGN2 protein, HMGN3a protein, HMGN3b protein, histone H1 centralglobular domain, imitation switch (ISWI) protein DNA binding domain,chromodomain-helicase-DNA protein 1 (CHD1) DNA binding domain, or acombination thereof.

The at least one nucleosome interacting protein domain of the fusionprotein used in the method can be linked to the CRISPR protein directlyvia a chemical bond, indirectly via a linker, or a combination thereof.The at least one nucleosome interacting protein domain can be linked tothe N-terminus, C-terminus, and/or an internal location of the CRISPRprotein. In some embodiments, the fusion protein used in the methodcomprises at least two nucleosome interacting protein domains linked tothe CRISPR protein.

The fusion proteins used in the methods disclosed herein can furthercomprise at least one nuclear localization signal, at least onecell-penetrating domain, at least one marker domain, or a combinationthereof.

In embodiments in which the method comprises introducing nucleic acidencoding the at least one fusion protein, the nucleic acid can be codonoptimized for translation in the eukaryotic cell. In some embodiments,the nucleic acids can be part of a vector such as, for example, a viralvector, a plasmid vector, or a self-replicating RNA.

In certain embodiments, the method can further comprise introducing intothe eukaryotic cell at least one donor polynucleotide, the donorpolynucleotide comprising at least one donor sequence.

The eukaryotic cells used in the methods disclosed herein can bemammalian cells. In some embodiments, the cells can be human cell. Thecells can be in vitro or in vivo.

Other aspects and features of the disclosure are detailed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the cleavage efficiency (as the percent of indels) ofwild-type CjCas9 (CjeCas9), a fusion protein comprising CjCas9 linked toHMGN1 and HMGB1 box A (CjeCas9-HN1HB1), and a fusion protein comprisingCjCas9 linked to HMGN1 and Histone H1 central globular domain(CjeCas9-HN1H1G) in the presence of wild-type sgRNA scaffold or modifiedsgRNA scaffold.

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for increasingthe accessibility of chromosomal DNA to programmable DNA modificationproteins including CRISPR systems. In particular, the present disclosureprovides fusion proteins comprising at least one nucleosome interactingprotein domain linked to a programmable DNA modification protein. Thenucleosome interacting protein domains alter or remodel nucleosomaland/or chromatin structure such that the programmable DNA modificationprotein has increased access to targeted chromosomal sequences, therebyincreasing the efficiency of targeted genome modification, targetedtranscriptional regulation, or targeted epigenetic modification.

(I) Fusion Proteins

One aspect of the present disclosure provides fusion proteins, whereineach fusion protein comprises at least one nucleosome interactingprotein domain linked to a programmable DNA modification protein. Theprogrammable DNA modification protein can have nuclease activity (seesection (I)(b)(i), below) or non-nuclease activity (see section(I)(b)(ii), below). Nucleosome interacting protein domains are describedbelow in section (I)(a) and linkages between the domains are describedbelow in section (I)(c).

(a) Nucleosome Interacting Protein Domains

Nucleosome interacting protein domains refer to chromosomal proteins orfragments thereof that interact with nucleosome and/or chromosomalproteins to facilitate nucleosome rearrangement and/or chromatinremodeling. In some embodiments, the nucleosome interacting proteindomain can be derived from high mobility group (HMG) box (HMGB)proteins. In other embodiments, the nucleosome interacting proteindomain can be HMG nucleosome-binding (HMGN) proteins or fragmentsthereof. In further embodiments, the nucleosome interacting proteindomain can be derived from linker histone H1 variants. In still otherembodiments, the nucleosome interacting protein domain can be derivedfrom chromatin remodeling complex proteins.

(i) HMGB Proteins

In some embodiments, the at least one nucleosome interacting proteindomain can be derived from an HMGB protein. HMGB proteins interact withnucleosomes and other chromosomal proteins to regulate chromatinstructure and function. Suitable HMGB proteins include mammalian HMGB1,mammalian HMGB2, and mammalian HMGB3. For example, the nucleosomeinteracting protein domain can be derived from a human HMGB1(RefSeqGene, U51677), human HMGB2 (RefSeqGene, M83665), or human HMGB3(RefSeqGene, NM_005342). In other embodiments, the nucleosomeinteracting protein domain can be derived from an HMGB protein orHMGB-like protein from other vertebrates, invertebrates (e.g.,Drosophila DSP1), plants, yeast, or other single cell eukaryotes.

In specific embodiments, the at least one nucleosome interacting proteindomain can be a fragment of an HMGB protein. In particular, the fragmentof the HMGB protein is a DNA-binding domain. HMGB proteins typicallycontain two DNA-binding domains, which are called box A and box B. Insome embodiments, the nucleosome interacting domain can be a box Adomain or a box B domain from a HMGB protein. In specific embodiments,the nucleosome interacting domain can be a HMGB1 box A domain, a HMGB2box A domain, or a HMGB3 box A domain.

(ii) HMGN Proteins

In other embodiments, the at least one nucleosome interacting proteindomain can be a HMGN protein or fragment thereof. HMGN proteins arechromosomal proteins that modulate the structure and function ofchromatin. Suitable mammalian HMGN proteins include HMGN1, HMGN2, HMGN3,HMGN3, HMGN4, and HMGN5. In various embodiments, the nucleosomeinteracting protein domain can be human HMGN1 (RefSeqGene, M21339),human HMGN2 (RefSeqGene, X13546), human HMGN3a or human HMGN3b(RefSeqGene, L40357), human HMGN4 (RefSeqGene, NM_030763), human HMGN5(RefSeqGene, NM_016710), a fragment thereof, or a derivative thereof. Inother embodiments, the nucleosome interacting protein domain can be anon-human HMGN protein, fragment, or derivative thereof. HMGN proteinsare relatively small proteins. As such, the entire HMGN protein can belinked to the programmable DNA modification protein. In someembodiments, however, a fragment (e.g., the centrally-locatednucleosome-binding domain) of a HMGN protein can be linked to theprogrammable DNA modification protein.

(iii) Histone H1 Variants

In still other embodiments, the at least one nucleosome interactingprotein domain can be derived from a linker histone H1 variant. Forexample, the nucleosome interacting protein domain can be a centralglobular domain from a histone H1 variant. Histone H1 variants bind tothe linker DNA between nucleosomes and the central globular domain (ofabout 80 amino acids) binds to the linker DNA at the nucleosome entryand exit sites close to the nucleosome dyad. Histone H1 variantscomprise a large family of related proteins with distinct specificityfor tissues, developmental stages, and organisms in which they areexpressed. For example, human and mouse contain 11 histone H1 variants,chicken has six variants (which are called histone H5), frog has fivevariants, nematode has eight variants, fruit fly species have from oneto three variants, and tobacco has six variants. In some embodiments,the histone H1 variant can be a human variant as shown below.

Protein name* Gene Symbol UniProtKB Accession Histone H1.0 H1F0 P07305Histone H1.1 HIST1H1A Q02539 Histone H1.2 HIST1H1C P16403 Histone H1.3HIST1H1D P16402 Histone H1.4 HIST1H1E P10412 Histone H1.5 HIST1H1BP16401 Histone H1.6 (testis specific) HIST1H1T P22492 Histone H1.7(testis specific) H1FNT Q75WM6 Histone H1.8 (oocyte specific) H1FOOQ8IZA3 Histone H1.9 (testis specific) HILS1 P60008 Histone H1.10 H1FXQ92522 *Talbert etal., Epigenetics & Chromatin, 2012, 5:7.

(iv) Chromatin Remodeling Complex Proteins

In further embodiments, the at least one nucleosome interacting proteindomain can be derived from a chromatin remodeling complex protein. Forexample, the nucleosome interacting protein domain can be DNA bindingdomain from a chromatin remodeling complex protein. Chromatin remodelingcomplexes are multi-subunit enzyme complexes with the capacity toremodel the structure of chromatin. These remodeling complexes use theenergy of ATP hydrolysis to move, destabilize, eject, or restructurenucleosomes.

Examples of chromatin remodeling complexes include SWI/SNF(SWitch/Sucrose Non-Fermentable), ISWI (Imitation SWitch), CHD(Chromodomain-Helicase-DNA binding), Mi-2/NuRD (Nucleosome Remodelingand Deacetylase), IN080, SWR1, and RSC complexes. In variousembodiments, the nucleosome interacting protein domain can be derivedfrom an ATPase, a helicase, and/or a DNA binding protein in thechromatin remodeling complex. In some embodiments, the nucleosomeinteracting protein domain can be derived from the ATPase ISWI from theISWI complex, the DNA-binding protein CHD1 from the CHD complex, theATP-dependent helicase SMARCA4 or the ATPase Snf2 from the SWI/SNFcomplex, ATPase Mi-2a or ATPase Mi2-3 of the Mi-1/NuRD complex, theRuvB-like AAA ATPase 1 or the RuvB-like AAA ATPase 2 from the IN080complex, the ATPase Swrl from the SWR1 complex, or the ATPase Rscl orATPase Rcs2 from the RSC complex. In specific embodiments, thenucleosome interacting protein domain can be a DNA binding domain fromISWI protein or a DNA binding domain from CHD1 protein.

(b) Programmable DNA Modification Proteins

A programmable DNA modification protein is a protein targeted to bind aspecific target sequence in chromosomal DNA, where it modifies the DNAor a protein associated with the DNA at or near the targeted sequence.Thus, a programmable DNA modification protein comprises a programmableDNA binding domain and a catalytically active modification domain.

The DNA binding domain of the programmable DNA modification protein isprogrammable, meaning that it can be designed or engineered to recognizeand bind different DNA sequences. In some embodiments, for example, DNAbinding is mediated by interactions between the DNA modification proteinand the target DNA. Thus, the DNA binding domain can be programmed tobind a DNA sequence of interest by protein engineering. In otherembodiments, for example, DNA binding is mediated by a guide RNA thatinteracts with the DNA modification protein and the target DNA. In suchinstances, the programmable DNA binding protein can be targeted to a DNAsequence of interest by designing the appropriate guide RNA.

A variety of modification domains can be included in the programmableDNA modification protein. In some embodiments, the modification domainhas nuclease activity and can cleave one or both strands of adouble-stranded DNA sequence. The DNA break can then be repaired by acellular DNA repair process such as non-homologous end-joining (NHEJ) orhomology-directed repair (HDR), such that the DNA sequence can bemodified by a deletion, insertion, and/or substitution of at least onebase pair. Examples of programmable DNA modification proteins havingnuclease activity include, without limit, CRISPR nucleases (ornickases), zinc finger nucleases, transcription activator-like effectornucleases, meganucleases, and a programmable DNA binding domain linkedto a nuclease domain. Programmable DNA modification proteins havingnuclease activity are detailed below in section (I)(b)(i).

In other embodiments, the modification domain of the programmable DNAmodification protein has non-nuclease activity (e.g., epigeneticmodification activity or transcriptional regulation activity) such thatthe programmable DNA modification protein modifies the structure and/oractivity of the DNA and/or protein(s) associated with the DNA. Thus, theprogrammable DNA modification protein can comprise a programmable DNAbinding domain linked to a non-nuclease domain. Such proteins aredetailed below in section (I)(b)(ii).

The programmable DNA modification proteins can comprise wild-type ornaturally-occurring DNA binding and/or modification domains, modifiedversions of naturally-occurring DNA binding and/or modification domains,synthetic or artificial DNA binding and/or modification domains, andcombinations thereof.

(i) Programmable DNA Modification Proteins with Nuclease Activity

Examples of programmable DNA modification proteins having nucleaseactivity include, without limit, CRISPR nucleases, zinc fingernucleases, transcription activator-like effector nucleases,meganucleases, and programmable DNA binding domains linked to nucleasedomains.

CRISPR Nucleases. The CRISPR nuclease can be derived from a type I, typeII (i.e., Cas9), type III, type V (i.e., Cpf1), or type VI (i.e., Cas13)CRISPR protein, which are present in various bacteria and archaea. Infurther embodiments, the CRISPR nuclease can be derived from an archaealCRISPR system, a CRISPR/CasX system, or a CRISPR/CasY system (Bursteinet al., Nature, 2017, 542(7640):237-241). In various embodiments, theCRISPR nuclease can be from Streptococcus sp. (e.g., S. pyogenes, S.thermophilus, S. pasteurianus), Campylobacter sp. (e.g., Campylobacterjejuni), Francisella sp. (e.g., Francisella novicida), Acaryochlorissp., Acetohalobium sp., Acidaminococcus sp., Acidithiobacillus sp.,Alicyclobacillus sp., Allochromatium sp., Ammonifex sp., Anabaena sp.,Arthrospira sp., Bacillus sp., Burkholderiales sp., Caldicelulosiruptorsp., Candidatus sp., Clostridium sp., Crocosphaera sp., Cyanothece sp.,Exiguobacterium sp., Finegoldia sp., Ktedonobacter sp., Lachnospiraceaesp., Lactobacillus sp., Lyngbya sp., Marinobacter sp., Methanohalobiumsp., Microscilla sp., Microcoleus sp., Microcystis sp., Natranaerobiussp., Neisseria sp., Nitrosococcus sp., Nocardiopsis sp., Nodularia sp.,Nostoc sp., Oscillatoria sp., Polaromonas sp., Pelotomaculum sp.,Pseudoalteromonas sp., Petrotoga sp., Prevotella sp., Staphylococcussp., Streptomyces sp., Streptosporangium sp., Synechococcus sp.,Thermosipho sp., or Verrucomicrobia sp.

The CRISPR nuclease can be a wild type or naturally-occurring protein.Alternatively, the CRISPR nuclease can be engineered to have improvedspecificity, altered PAM specificity, decreased off-target effects,increased stability, and the like.

In some embodiments, the CRISPR nuclease can be a type II CRISPR/Cas 9protein. For example, the CRISPR nuclease can be Streptococcus pyogenesCas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcuspasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9),Staphylococcus aureus (SaCas9), Francisella novicida Cas9 (FnCas9),Neisseria cinerea Cas9 (NcCas9), or Neisseria meningitis Cas9 (NmCas9).In other embodiments, the CRISPR nuclease can be a type V CRISPR/Cpf1protein, e.g., Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp.Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). Infurther embodiments, the CRISPR nuclease can be a type VI CRISPR/Cas13protein, e.g., Leptotrichia wadei Cas13a (LwaCas13a) or Leptotrichiashahii Cas13a (LshCas13a).

In general, the CRISPR nuclease comprises at least one nuclease domainhaving endonuclease activity. For example, a Cas9 nuclease comprises aHNH domain, which cleaves the guide RNA complementary strand, and a RuvCdomain, which cleaves the non-complementary strand, a Cpf1 proteincomprises a RuvC domain and a NUC domain, and a Cas13a nucleasecomprises two HNEPN domains. In some embodiments, both nuclease domainsare active and the CRISPR nuclease has double-stranded cleavage activity(i.e., cleaves both strands of a double-stranded nucleic acid sequence).In other embodiments, one of the nuclease domains is inactivated by oneor more mutations and/or deletions, and the CRISPR variant is a nickasethat cleaves one strand of a double-stranded nucleic acid sequence. Forexample, one or more mutations in the RuvC domain of Cas9 protein (e.g.,D10A, D8A, E762A, and/or D986A) results in an HNH nickase that nicks theguide RNA complementary strand; and one or more mutations in the HNHdomain of Cas9 protein (e.g., H840A, H559A, N854A, N856A, and/or N863A)results in a RuvC nickase that nicks the guide RNA non-complementarystrand. Comparable mutations can convert Cpf1 and Cas13a nucleases tonickases.

Zinc Finger Nucleases.

In still other embodiments, the programmable DNA modification proteinhaving nuclease activity can be a pair of zinc finger nucleases (ZFN). AZFN comprises a DNA binding zinc finger region and a nuclease domain.The zinc finger region can comprise from about two to seven zincfingers, for example, about four to six zinc fingers, wherein each zincfinger binds three consecutive base pairs. The zinc finger region can beengineered to recognize and bind to any DNA sequence. Zinc finger designtools or algorithms are available on the internet or from commercialsources. The zinc fingers can be linked together using suitable linkersequences.

A ZFN also comprises a nuclease domain, which can be obtained from anyendonuclease or exonuclease. Non-limiting examples of endonucleases fromwhich a nuclease domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. In some embodiments,the nuclease domain can be derived from a type II-S restrictionendonuclease. Type II-S endonucleases cleave DNA at sites that aretypically several base pairs away from the recognition/binding site and,as such, have separable binding and cleavage domains. These enzymesgenerally are monomers that transiently associate to form dimers tocleave each strand of DNA at staggered locations. Non-limiting examplesof suitable type II-S endonucleases include Bfil, Bpml, Bsal, Bsgl,BsmBI, Bsml, BspMI, FokI, Mboll, and Sapl. In some embodiments, thenuclease domain can be a FokI nuclease domain or a derivative thereof.The type II-S nuclease domain can be modified to facilitate dimerizationof two different nuclease domains. For example, the cleavage domain ofFokI can be modified by mutating certain amino acid residues. By way ofnon-limiting example, amino acid residues at positions 446, 447, 479,483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538of FokI nuclease domains are targets for modification. In specificembodiments, the FokI nuclease domain can comprise a first FokIhalf-domain comprising Q486E, 1499L, and/or N496D mutations, and asecond FokI half-domain comprising E490K, 1538K, and/or H537R mutations.In some embodiments, the ZFN has double-stranded cleavage activity. Inother embodiments, the ZFN has nickase activity (i.e., one of thenuclease domains has been inactivated).

Transcription Activator-Like Effector Nucleases.

In alternate embodiments, the programmable DNA modification proteinhaving nuclease activity can be a transcription activator-like effectornuclease (TALEN). TALENs comprise a DNA binding domain composed ofhighly conserved repeats derived from transcription activator-likeeffectors (TALEs) that is linked to a nuclease domain. TALEs areproteins secreted by plant pathogen Xanthomonas to alter transcriptionof genes in host plant cells. TALE repeat arrays can be engineered viamodular protein design to target any DNA sequence of interest. Thenuclease domain of TALENs can be any nuclease domain as described abovein the subsection describing ZFNs. In specific embodiments, the nucleasedomain is derived from FokI (Sanjana et al., 2012, Nat Protoc,7(1):171-192). The TALEN can have double-stranded cleavage activity ornickase activity.

Meganucleases or Rare-Cutting Endonucleases.

In still other embodiments, the programmable DNA modification proteinhaving nuclease activity can be a meganuclease or derivative thereof.Meganucleases are endodeoxyribonucleases characterized by longrecognition sequences, i.e., the recognition sequence generally rangesfrom about 12 base pairs to about 45 base pairs. As a consequence ofthis requirement, the recognition sequence generally occurs only once inany given genome. Among meganucleases, the family of homingendonucleases named LAGLIDADG has become a valuable tool for the studyof genomes and genome engineering. In some embodiments, the meganucleasecan be I-Scel, I-Tevl, or variants thereof. A meganuclease can betargeted to a specific chromosomal sequence by modifying its recognitionsequence using techniques well known to those skilled in the art. Inalternate embodiments, the programmable DNA modification protein havingnuclease activity can be a rare-cutting endonuclease or derivativethereof. Rare-cutting endonucleases are site-specific endonucleaseswhose recognition sequence occurs rarely in a genome, preferably onlyonce in a genome. The rare-cutting endonuclease may recognize a7-nucleotide sequence, an 8-nucleotide sequence, or longer recognitionsequence. Non-limiting examples of rare-cutting endonucleases includeNotI, AscI, PacI, AsiSI, SbfI, and FseI.

Programmable DNA Binding Domains Linked to Nuclease Domains.

In yet additional embodiments, the programmable DNA modification proteinhaving nuclease activity can be a chimeric protein comprising aprogrammable DNA binding domain linked to a nuclease domain. Thenuclease domain can be any of those described above in the subsectiondescribing ZFNs (e.g., the nuclease domain can be a FokI nucleasedomain), a nuclease domain derived from a CRISPR nuclease (e.g., RuvC orHNH nuclease domains of Cas9), or a nuclease domain derived from ameganuclease or rare-cutting endonuclease.

The programmable DNA binding domain of the chimeric protein can be anyprogrammable DNA binding protein such as, e.g., a zinc finger protein ora transcription activator-like effector. Alternatively, the programmableDNA binding domain can be a catalytically inactive (dead) CRISPR proteinthat was modified by deletion or mutation to lack all nuclease activity.For example, the catalytically inactive CRISPR protein can be acatalytically inactive (dead) Cas9 (dCas9) in which the RuvC domaincomprises a D10A, D8A, E762A, and/or D986A mutation and the HNH domaincomprises a H840A, H559A, N854A, N865A, and/or N863A mutation.Alternatively, the catalytically inactive CRISPR protein can be acatalytically inactive (dead) Cpf1 protein comprising comparablemutations in the nuclease domains. In still other embodiments, theprogrammable DNA binding domain can be a catalytically inactivemeganuclease in which nuclease activity was eliminated by mutationand/or deletion, e.g., the catalytically inactive meganuclease cancomprise a C-terminal truncation.

(ii) Programmable DNA Modification Proteins with Non-Nuclease Activity

In alternate embodiments, the programmable DNA modification protein canbe a chimeric protein comprising a programmable DNA binding domainlinked to a non-nuclease domain. The programmable DNA binding domain canbe a zinc finger protein, a transcription activator-like effector, acatalytically inactive (dead) CRISPR protein, or a catalyticallyinactive (dead) meganuclease. For example, the catalytically inactiveCRISPR protein can be a catalytically inactive (dead) Cas9 (dCas9) inwhich the RuvC domain comprises a D10A, D8A, E762A, and/or D986Amutation and the HNH domain comprises a H840A, H559A, N854A, N865A,and/or N863A mutation. Alternatively, the catalytically inactive CRISPRprotein can be a catalytically inactive (dead) Cpf1 protein comprisingcomparable mutations in the nuclease domains.

In some embodiments, the non-nuclease domain of the chimeric protein canbe an epigenetic modification domain, which alters DNA or chromatinstructure (and may or may not alter DNA sequence). Non-limiting examplesof suitable epigenetic modification domains include those with DNAmethyltransferase activity (e.g., cytosine methyltransferase), DNAdemethylase activity, DNA deamination (e.g., cytosine deaminase,adenosine deaminase, guanine deaminase), DNA amination, DNA oxidationactivity, DNA helicase activity, histone acetyltransferase (HAT)activity (e.g., HAT domain derived from E1A binding protein p300),histone deacetylase activity, histone methyltransferase activity,histone demethylase activity, histone kinase activity, histonephosphatase activity, histone ubiquitin ligase activity, histonedeubiquitinating activity, histone adenylation activity, histonedeadenylation activity, histone SUMOylating activity, histonedeSUMOylating activity, histone ribosylation activity, histonederibosylation activity, histone myristoylation activity, histonedemyristoylation activity, histone citrullination activity, histonealkylation activity, histone dealkylation activity, or histone oxidationactivity. In specific embodiments, the epigenetic modification domaincan comprise cytidine deaminase activity, histone acetyltransferaseactivity, or DNA methyltransferase activity.

In other embodiments, the non-nuclease modification domain of thechimeric protein can be a transcriptional activation domain ortranscriptional repressor domain. Suitable transcriptional activationdomains include, without limit, herpes simplex virus VP16 domain, VP64(which is a tetrameric derivative of VP16), VP160, NFκB p65 activationdomains, p53 activation domains 1 and 2, CREB (cAMP response elementbinding protein) activation domains, E2A activation domains, activationdomain from human heat-shock factor 1 (HSF1), or NFAT (nuclear factor ofactivated T-cells) activation domains. Non-limiting examples of suitabletranscriptional repressor domains include inducible cAMP early repressor(ICER) domains, Kruppel-associated box (KRAB) repressor domains, YY1glycine rich repressor domains, Spl-like repressors, E(spl) repressors,IκB repressor, or methyl-CpG binding protein 2 (MeCP2) repressor.Transcriptional activation or transcriptional repressor domains can begenetically fused to the DNA binding protein or bound via noncovalentprotein-protein, protein-RNA, or protein-DNA interactions.

In particular embodiments, the non-nuclease domain of the chimericprotein can comprise cytidine deaminase activity, histoneacetyltransferase activity, transcriptional activation activity, ortranscriptional repressor activity.

In some embodiments, the chimeric protein having non-nuclease activitycan further comprise at least one detectable label. The detectable labelcan be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green,Alexa Fluors, Halo tags, or suitable fluorescent dye), a detection tag(e.g., biotin, digoxigenin, and the like), quantum dots, or goldparticles.

(c) Linkages

The fusion proteins disclosed herein comprise at least one nucleosomeinteracting protein domain linked to a programmable DNA modificationprotein. The linkage between the at least one nucleosome interactingprotein domain and the programmable DNA modification protein can bedirect via a chemical bond, or the linkage can be indirect via a linker.

In some embodiments, the at least one nucleosome interacting proteindomain can be linked directly to the programmable DNA modificationprotein by a covalent bond (e.g., peptide bond, ester bond, and thelike). Alternatively, the chemical bond can be non-covalent (e.g.,ionic, electrostatic, hydrogen, hydrophobic, Van der interactions, ornr-effects).

In other embodiments, the at least one nucleosome interacting proteindomain can be linked to the programmable DNA modification protein by alinker. A linker is a chemical group that connects one or more otherchemical groups via at least one covalent bond. Suitable linkers includeamino acids, peptides, nucleotides, nucleic acids, organic linkermolecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole,biphenyl-3,4′,5-tricarboxylic acid, p-am inobenzyloxycarbonyl, and thelike), disulfide linkers, and polymer linkers (e.g., PEG). The linkercan include one or more spacing groups including, but not limited toalkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl,heteroaryl, aralkyl, aralkenyl, aralkynyl and the like. The linker canbe neutral, or carry a positive or negative charge. Additionally, thelinker can be cleavable such that the linker's covalent bond thatconnects the linker to another chemical group can be broken or cleavedunder certain conditions, including pH, temperature, salt concentration,light, a catalyst, or an enzyme.

In still other embodiments, the at least one nucleosome interactingprotein domain can be linked to the programmable DNA modificationprotein by peptide linkers. The peptide linker can be a flexible aminoacid linker (e.g., comprising small, non-polar or polar amino acids).Non-limiting examples of flexible linkers include LEGGGS (SEQ ID NO:1),TGSG (SEQ ID NO:2), GGSGGGSG (SEQ ID NO:3), (GGGGS)₁₋₄ (SEQ ID NO:4),and (Gly)₆₋₈ (SEQ ID NO:5). Alternatively, the peptide linker can be arigid amino acid linker. Such linkers include (EAAAK)₁₋₄ (SEQ ID NO:6),A(EAAAK)₂₋₅A (SEQ ID NO:7), PAPAP (SEQ ID NO:8), and (AP)₆₋₈ (SEQ IDNO:9). Examples of suitable linkers are well known in the art andprograms to design linkers are readily available (Crasto et al., ProteinEng., 2000, 13(5):309-312).

The at least one nucleosome interacting protein domain can be linked toN-terminus, the C-terminus, and/or an internal location of theprogrammable DNA modification protein. In some embodiments, at least onenucleosome interacting protein domain can be linked to N-terminus of theprogrammable DNA modification protein. In other embodiments, the atleast one nucleosome interacting protein domain can be linked toC-terminus of the programmable DNA modification protein. In still otherembodiments, at least one nucleosome interacting protein domain can belinked to N-terminus and at least one nucleosome interacting proteindomain can be linked to C-terminus of the programmable DNA modificationprotein.

In some embodiments, the fusion protein can comprise one nucleosomeinteracting protein domain. In other embodiments, the fusion protein cancomprise two nucleosome interacting protein domains. In still otherembodiments, the fusion protein can comprise three nucleosomeinteracting protein domains. In additional embodiments, the fusionprotein can comprise four, five, or more than five nucleosomeinteracting protein domains. The one or more nucleosome interactingprotein domains can be the same or they can be different.

In embodiments in which the fusion protein comprises two or morenucleosome interacting protein domains, the two or more nucleosomeinteracting domains can be linked to either end, both ends, and/or aninternal location of the programmable DNA modification protein. The twoor more nucleosome interacting protein domains can be the same or theycan be different. For example, the complex can comprise at least two HMGDNA-binding domains, at least two HMGN proteins, at least one HMGDNA-binding domain and at least one HMGN protein, at least one HMGDNA-binding domain or HMGN protein and at least one central domain froma histone H1 variant, at least one HMG DNA-binding domain or HMGNprotein and at least one domain from a chromatin remodeling complexprotein, at least one HMG DNA-binding domain or HMGN protein, at leastone histone H1 variant central domain, and at least one domain from achromatin remodeling complex protein, and the like.

(d) Optional Nuclear Localization Signal, Cell-Penetrating Domain,and/or Marker Domain

The fusion proteins disclosed herein can further comprise at least onenuclear localization signal, cell-penetrating domain, and/or markerdomain.

Non-limiting examples of nuclear localization signals include PKKKRKV(SEQ ID NO:10), PKKKRRV (SEQ ID NO:11), KRPAATKKAGQAKKKK (SEQ ID NO:12),YGRKKRRQRRR (SEQ ID NO:13), RKKRRQRRR (SEQ ID NO:14), PAAKRVKLD (SEQ IDNO:15), RQRRNELKRSP (SEQ ID NO:16), VSRKRPRP (SEQ ID NO:17), PPKKARED(SEQ ID NO:18), PQPKKKPL (SEQ ID NO:19), SALIKKKKKMAP (SEQ ID NO:20),PKQKKRK (SEQ ID NO:21), RKLKKKIKKL (SEQ ID NO:22), REKKKFLKRR (SEQ IDNO:23), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:24), RKCLQAGMNLEARKTKK (SEQ IDNO:25), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:26), andRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:27).

Examples of suitable cell-penetrating domains include, without limit,GRKKRRQRRRPPQPKKKRKV (SEQ ID NO:28), PLSSIFSRIGDPPKKKRKV (SEQ ID NO:29),GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO:30), GALFLGFLGAAGSTMGAWSQPKKKRKV(SEQ ID NO:31), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:32), YARAAARQARA (SEQID NO:33), THRLPRRRRRR (SEQ ID NO:34), GGRRARRRRRR (SEQ ID NO:35),RRQRRTSKLMKR (SEQ ID NO:36), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:37),KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:38), and RQIKIWFQNRRMKWKK(SEQ ID NO:39).

Marker domains include fluorescent proteins and purification or epitopetags. Suitable fluorescent proteins include, without limit, greenfluorescent proteins (e.g., GFP, eGFP, GFP-2, tagGFP, turboGFP, Emerald,Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellowfluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP,ZsYellowl), blue fluorescent proteins (e.g., BFP, EBFP, EBFP2, Azurite,mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g.,ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescentproteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins(e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,mTangerine, tdTomato). Non-limiting examples of suitable purification orepitope tags include 6×His, FLAG®, HA, GST, Myc, and the like.

The at least one nuclear localization signal, cell-penetrating domain,and/or marker domain can be located at the N-terminus, the C-terminus,and/or in an internal location of the fusion protein.

(e) Specific Fusion Proteins

In general, the at least one nucleosome interacting protein domain ofthe fusion protein is chosen from HMGB1 box A domain, HMGN1 protein,HMGN2 protein, HMGN3a protein, HMGN3b protein, histone H1 centralglobular domain, imitation switch (ISWI) protein DNA binding domain,chromodomain-helicase-DNA protein 1 (CHD1) DNA binding domain, orcombinations thereof.

In specific embodiments, the programmable DNA modification protein ofthe fusion protein is a CRISPR protein. For example, the CRISPR proteincan be Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilusCas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacterjejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9), Francisellanovicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), Neisseriameningitis Cas9 (NmCas9), Francisella novicida Cpf1 (FnCpf1),Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006Cpf1 (LbCpf1).

In some embodiments, the fusion protein has an amino acid sequencehaving at least about 80% sequence identity with any of SEQ IDNOS:61-79. In general, any amino acid substitution is conservative,i.e., limited to exchanges within members of group 1: glycine, alanine,valine, leucine, and Isoleucine; group 2: serine, cysteine, threonine,and methionine; group 3: proline; group 4: phenylalanine, tyrosine, andtryptophan; and group 5: aspartate, glutamate, asparagine, andglutamine. In various embodiments, the amino acid sequence of the fusionprotein has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% sequence identitywith any of SEQ ID NOS:61-79. In some embodiments, the fusion proteinhas an amino acid sequence as set forth in SEQ ID NO:61, SEQ ID NO:62,SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77,SEQ ID NO:78, or SEQ ID NO:79.

(II) Complexes

Another aspect of the present disclosure encompasses complexescomprising at least one CRISPR system (i.e., CRISPR protein and guideRNA) and at least one nucleosome interacting protein domain. In someembodiments, the at least one nucleosome interacting protein domain canbe linked to the CRISPR protein of the CRISPR system (i.e., the complexcomprises a CRISPR fusion protein as described in section (I) above). Inother embodiments, the at least one nucleosome interacting proteindomain can be linked to the guide RNA of the CRISPR system. The linkagecan be direct or indirect, essentially as described above in section(I)(c). For example, a nucleosome interacting protein domain can belinked to an RNA aptamer binding protein, and the guide RNA can compriseaptamer sequences, such that binding of the RNA aptamer binding proteinto the RNA aptamer sequence links the nucleosome interacting proteindomain to the guide RNA.

Nucleosome interacting protein domains are described above in section(I)(a), and CRISPR proteins are detailed above in section (I)(b). TheCRISPR protein can have nuclease or nickase activity (e.g., can be atype II CRISPR/Cas9, type V CRISPR/Cpf1, or type VI CRISPR/Cas13). Forexample, a complex can comprise a CRISPR nuclease, or a complex cancomprise two CRISPR nickases. Alternatively, the CRISPR protein can bemodified to lack all nuclease activity and linked to non-nucleasedomains (e.g., domains having cytosine deaminase activity, histoneacetyltransferase activity, transcriptional activation activity, ortranscriptional repressor activity). In some embodiments, thenon-nuclease domain also can be linked to an RNA aptamer bindingprotein.

A guide RNA comprises (i) a CRISPR RNA (crRNA) that contains a guidesequence at the 5′ end that hybridizes with a target sequence and (ii) atransacting crRNA (tracrRNA) sequence that interacts with the CRISPRprotein. The crRNA guide sequence of each guide RNA is different (i.e.,is sequence specific). The tracrRNA sequence is generally the same inguide RNAs designed to complex with a CRISPR protein from a particularbacterial species.

The crRNA guide sequence is designed to hybridize with a target sequence(i.e., protospacer) that is bordered by a protospacer adjacent motif(PAM) in a double-stranded sequence. PAM sequences for Cas9 proteinsinclude 5′-NGG, 5′-NGGNG, 5′-NNAGAAW, and 5′-ACAY, and PAM sequences forCpf1 include 5′-TTN (wherein N is defined as any nucleotide, W isdefined as either A or T, and Y is defined as either C or T). Ingeneral, the complementarity between the crRNA guide sequence and thetarget sequence is at least 80%, at least 85%, at least 90%, at least95%, or at least 99%. In specific embodiments, the complementarity iscomplete (i.e., 100%). In various embodiments, the length of the crRNAguide sequence can range from about 15 nucleotides to about 25nucleotides. For example, the crRNA guide sequence can be about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In specificembodiments, the crRNA can be about 19, 20, 21, or 22 nucleotides inlength.

The crRNA and tracrRNA comprise repeat sequences that form one or moreone-stem loop structures, which can interact with the CRISPR protein.The length of each loop and stem can vary. For example, the one or moreloops can range from about 3 to about 10 nucleotides in length, and theone or more stems can range from about 6 to about 20 base pairs inlength. The one or more stems can comprise one or more bulges of 1 toabout 10 nucleotides.

The crRNA can range in length from about 25 nucleotides to about 100nucleotides. In various embodiments, the crRNA can range in length fromabout 25 to about 50 nucleotides, from about 50 to about 75 nucleotides,or from about 75 to about 100 nucleotides. The tracrRNA can range inlength from about 50 nucleotides to about 300 nucleotides. In variousembodiments, the tracrRNA can range in length from about 50 to about 90nucleotides, from about 90 to about 110 nucleotides, from about 110 toabout 130 nucleotides, from about 130 to about 150 nucleotides, fromabout 150 to about 170 nucleotides, from about 170 to about 200nucleotides, from about 200 to about 250 nucleotides, or from about 250to about 300 nucleotides.

The tracrRNA sequence in the guide RNA generally is based upon thecoding sequence of wild type tracrRNA in the bacterial species ofinterest. In some embodiments, the wild-type tracrRNA sequence (or thecrRNA constant repeat region and the corresponding 5′ region of thetracrRNA that forms a duplex structure with the crRNA constant repeatregion) can be modified to facilitate secondary structure formation,increase secondary structure stability, facilitate expression ineukaryotic cells, increase editing efficiency, and so forth. Forexample, one or more nucleotide changes can be introduced into theconstant guide RNA sequence (see Example 8, below).

The guide RNA can be a single molecule (i.e., a single guide RNA orsgRNA), wherein the crRNA sequence is linked to the tracrRNA sequence.Alternatively, the guide RNA can be two separate molecules. A firstmolecule comprising the crRNA guide sequence at the 5′ end andadditional sequence at 3′ end that is capable of base pairing with the5′ end of a second molecule, wherein the second molecule comprises 5′sequence that is capable of base pairing with the 3′ end of the firstmolecule, as well as additional tracrRNA sequence. In some embodiments,the guide RNA of type V CRISPR/Cpf1 systems can comprise only crRNA.

In some embodiments, the one or more stem-loop regions of the guide RNAcan be modified to comprise one or more aptamer sequences (Konermann etal., Nature, 2015, 517(7536):583-588; Zalatan et al., Cell, 2015,160(1-2):339-50). Examples of suitable RNA aptamer protein domainsinclude MS2 coat protein (MCP), PP7 bacteriophage coat protein (PCP), Mubacteriophage Com protein, lambda bacteriophage N22 protein, stem-loopbinding protein (SLBP), Fragile X mental retardation syndrome-relatedprotein 1 (FXR1), proteins derived from bacteriophage such as AP205,BZ13, f1, f2, fd, fr, ID2, JP34/GA, JP501, JP34, JP500, KU1, M11, M12,MX1, NL95, PP7, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, Qβ, R17, SP-β, TW18, TW19,and VK, fragments thereof, or derivatives thereof. The length of theadditional aptamer sequence can range from about 20 nucleotides to about200 nucleotides.

The guide RNA can comprise standard ribonucleotides, modifiedribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and/orribonucleotide analogs. In some embodiments, the guide RNA can furthercomprise at least one detectable label. The detectable label can be afluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, AlexaFluors, Halo tags, or suitable fluorescent dye), a detection tag (e.g.,biotin, digoxigenin, and the like), quantum dots, or gold particles.Those skilled in the art are familiar with gRNA design and construction,e.g., gRNA design tools are available on the internet or from commercialsources.

The guide RNA can be synthesized chemically, synthesized enzymatically,or a combination thereof. For example, the guide RNA can be synthesizedusing standard phosphoramidite-based solid-phase synthesis methods.Alternatively, the guide RNA can be synthesized in vitro by operablylinking DNA encoding the guide RNA to a promoter control sequence thatis recognized by a phage RNA polymerase. Examples of suitable phagepromoter sequences include T7, T3, SP6 promoter sequences, or variationsthereof. In embodiments in which the guide RNA comprises two separatemolecules (i.e., crRNA and tracrRNA), the crRNA can be chemicallysynthesized and the tracrRNA can be enzymatically synthesized.

(III) Nucleic Acids

A further aspect of the present disclosure provides nucleic acidsencoding the fusion proteins described above in section (I) and theCRISPR complexes described in section (II). The CRISPR complexes can beencoded by single nucleic acids or multiple nucleic acids. The nucleicacids can be DNA or RNA, linear or circular, single-stranded ordouble-stranded. The RNA or DNA can be codon optimized for efficienttranslation into protein in the eukaryotic cell of interest. Codonoptimization programs are available as freeware or from commercialsources.

In some embodiments, the nucleic acid encoding the fusion protein or theprotein components of the CRISPR complex can be RNA. The RNA can beenzymatically synthesized in vitro. For this, DNA encoding the proteinof interest can be operably linked to a promoter sequence that isrecognized by a phage RNA polymerase for in vitro RNA synthesis. Forexample, the promoter sequence can be a T7, T3, or SP6 promoter sequenceor a variation of a T7, T3, or SP6 promoter sequence. The DNA encodingthe protein can be part of a vector, as detailed below. In suchembodiments, the in vitro-transcribed RNA can be purified, capped,and/or polyadenylated. In other embodiments, the RNA encoding the fusionprotein or protein component of the complex can be part of aself-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013,13:246-254). The self-replicating RNA can be derived from anoninfectious, self-replicating Venezuelan equine encephalitis (VEE)virus RNA replicon, which is a positive-sense, single-stranded RNA thatis capable of self-replicating for a limited number of cell divisions,and which can be modified to code proteins of interest (Yoshioka et al.,Cell Stem Cell, 2013, 13:246-254).

In other embodiments, the nucleic acid encoding the fusion protein orthe CRISPR protein and guide RNA can be DNA. The DNA coding sequence canbe operably linked to at least one promoter control sequence forexpression in the cell of interest. In certain embodiments, the DNAcoding sequence can be operably linked to a promoter sequence forexpression of the protein or RNA in bacterial (e.g., E. coli) cells oreukaryotic (e.g., yeast, insect, or mammalian) cells. Suitable bacterialpromoters include, without limit, T7 promoters, lac operon promoters,trp promoters, tac promoters (which are hybrids of trp and lacpromoters), variations of any of the foregoing, and combinations of anyof the foregoing. Non-limiting examples of suitable eukaryotic Pol IIpromoters include constitutive, regulated, or cell- or tissue-specificpromoters. Suitable eukaryotic constitutive promoter control sequencesinclude, but are not limited to, cytomegalovirus immediate earlypromoter (CMV), simian virus (SV40) promoter, adenovirus major latepromoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus(MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongationfactor (ED1)-alpha promoter, ubiquitin promoters, actin promoters,tubulin promoters, immunoglobulin promoters, fragments thereof, orcombinations of any of the foregoing. Examples of suitable eukaryoticregulated promoter control sequences include, without limit, thoseregulated by heat shock, metals, steroids, antibiotics, or alcohol.Non-limiting examples of tissue-specific promoters include B29 promoter,CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desminpromoter, elastase-1 promoter, endoglin promoter, fibronectin promoter,Fit-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-βpromoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter,SYN1 promoter, and WASP promoter. The promoter sequence can be wild typeor it can be modified for more efficient or efficacious expression. Insome embodiments, the DNA coding sequence also can be linked to apolyadenylation signal (e.g., SV40 polyA signal, bovine growth hormone(BGH) polyA signal, etc.) and/or at least one transcriptionaltermination sequence. The sequence encoding the guide RNA is operablylinked to a Pol III promoter control sequence for expression ineukaryotic cells. Examples of suitable Pol III promoters include, butare not limited to, mammalian U6, U3, H1, and 7SL RNA promoters. In somesituations, the fusion protein or components of the complex can bepurified from bacterial or eukaryotic cells.

In various embodiments, nucleic acid encoding the fusion protein or theCRISPR protein and guide RNA of the complex can be present in a vector.Suitable vectors include plasmid vectors, viral vectors, andself-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013,13:246-254). In some embodiments, the nucleic acid encoding the fusionprotein or the components of the complex can be present in a plasmidvector. Non-limiting examples of suitable plasmid vectors include pUC,pBR322, pET, pBluescript, and variants thereof. In other embodiments,the nucleic acid encoding the fusion protein or the components of thecomplex or can be part of a viral vector (e.g., lentiviral vectors,adeno-associated viral vectors, adenoviral vectors, and so forth). Theplasmid or viral vector can comprise additional expression controlsequences (e.g., enhancer sequences, Kozak sequences, polyadenylationsequences, transcriptional termination sequences, etc.), selectablemarker sequences (e.g., antibiotic resistance genes), origins ofreplication, and the like. Additional information about vectors and usethereof can be found in “Current Protocols in Molecular Biology” Ausubelet al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: ALaboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 3^(rd) edition, 2001.

(IV) Kits

A further aspect of the present disclosure provides kits comprising theat least one of the fusion proteins detailed above in section (I), atleast one of the CRISPR complexes described above in section (II),and/or at least one of the nucleic acids described above in section(III). The kits can further comprise transfection reagents, cell growthmedia, selection media, in-vitro transcription reagents, nucleic acidpurification reagents, protein purification reagents, buffers, and thelike. The kits provided herein generally include instructions forcarrying out the methods detailed below. Instructions included in thekits may be affixed to packaging material or may be included as apackage insert. While the instructions are typically written or printedmaterials, they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this disclosure. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. As used herein, theterm “instructions” can include the address of an internet site thatprovides the instructions.

(V) Cells

The present disclosure also provides cells comprising the at least oneof the fusion proteins detailed above in section (I), at least one ofthe CRISPR complexes described above in section (II), and/or at leastone of the nucleic acids described above in section (III). In general,the cell is a eukaryotic cell. For example, the cell can be a humancell, a non-human mammalian cell, a non-mammalian vertebrate cell, aninvertebrate cell, an insect cell, a plant cell, a yeast cell, or asingle cell eukaryotic organism.

(VI) Methods for Increasing Efficiency of Targeted Genome,Transcriptional, or Epigenetic Modification

Another aspect of the present disclosure encompasses methods forincreasing the efficiency of targeted genome modification, targetedtranscriptional modification, or targeted epigenetic modification ineukaryotic cells by increasing the accessibility of a programmable DNAmodification protein to its target sequence in chromosomal DNA. In someembodiments, the method comprises introducing into the eukaryotic cellof interest at least one of the fusion proteins described above insection (I), at least one of the CRISPR complexes described above insection (II), or nucleic acid encoding the at least one fusion proteinor CRISPR complex as described above in section (III), and optionally, adonor polynucleotide.

The programmable DNA modification protein of the fusion protein isengineered to recognize and bind to a target sequence in chromosomalDNA, and the one or more nucleosome interacting protein domains of thefusion protein interact with nucleosomes at or near the target sequenceto alter or remodel nucleosomal and/or chromatin structure. As aconsequence, the DNA modification protein has increased access to thetarget chromosomal sequence such that efficiency of modification by theDNA modification protein is increased. In specific embodiments, thefusion protein comprises at least one nucleosome interacting proteindomain linked to a CRISPR nuclease, such that interactions between thenucleosome interacting protein domain(s) and nucleosomes/chromatin at ornear the target sequence increases the efficiency to targeted genomicmodifications (see, Examples 1-8).

Thus, the methods disclosed herein can increase the efficiency oftargeted genome editing (e.g., gene corrections, gene knock-outs, geneknock-ins, and the like), targeted epigenetic modifications, andtargeted transcriptional regulation.

(a) Introduction into the Cell

As mentioned above, the method comprises introducing into the cell atleast one fusion protein, at least one CRISPR complex, or nucleicacid(s) encoding said fusion protein or CRISPR complex (and, optionally,a donor polynucleotide). The at least one fusion protein, CRISPRcomplex, or nucleic acid(s) can be introduced into the cell of interestby a variety of means.

In some embodiments, the cell can be transfected with the appropriatemolecules (i.e., protein, DNA, and/or RNA). Suitable transfectionmethods include nucleofection (or electroporation), calciumphosphate-mediated transfection, cationic polymer transfection (e.g.,DEAE-dextran or polyethylenimine), viral transduction, virosometransfection, virion transfection, liposome transfection, cationicliposome transfection, immunoliposome transfection, nonliposomal lipidtransfection, dendrimer transfection, heat shock transfection,magnetofection, lipofection, gene gun delivery, impalefection,sonoporation, optical transfection, and proprietary agent-enhanceduptake of nucleic acids. Transfection methods are well known in the art(see, e.g., “Current Protocols in Molecular Biology” Ausubel et al.,John Wiley & Sons, New York, 2003 or “Molecular Cloning: A LaboratoryManual” Sambrook & Russell, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 3rd edition, 2001). In other embodiments, the moleculescan be introduced into the cell by microinjection. For example, themolecules can be injected into the cytoplasm or nuclei of the cells ofinterest. The amount of each molecule introduced into the cell can vary,but those skilled in the art are familiar with means for determining theappropriate amount.

The various molecules can be introduced into the cell simultaneously orsequentially. For example, the fusion protein or CRISPR complex (orencoding nucleic acids) and the donor polynucleotide can be introducedat the same time. Alternatively, one can be introduced first and thenthe other can be introduced later into the cell.

In general, the cell is maintained under conditions appropriate for cellgrowth and/or maintenance. Suitable cell culture conditions are wellknown in the art and are described, for example, in Santiago et al.,Proc. Natl. Acad. Sci. USA, 2008, 105:5809-5814; Moehle et al. Proc.Natl. Acad. Sci. USA, 2007, 104:3055-3060; Urnov et al., Nature, 2005,435:646-651; and Lombardo et al., Nat. Biotechnol., 2007, 25:1298-1306.Those of skill in the art appreciate that methods for culturing cellsare known in the art and can and will vary depending on the cell type.Routine optimization may be used, in all cases, to determine the besttechniques for a particular cell type.

(b) Targeted Genome or Epigenetic Modification

The one or more nucleosome interacting protein domains of the fusionprotein or CRISPR complex interacts with nucleosomes and/or chromosomalDNA at or near the target chromosomal sequence such that nucleosomaland/or chromatin structure is altered/remodeled, thereby increasingaccessibility of the programmable DNA modification protein of the fusionprotein or the CRISPR protein of the CRISPR complex to the targetchromosomal sequence. Increased access to the target chromosomalsequence results in increased frequency/efficiency of targeted genome,transcriptional, or epigenetic modification.

In embodiments in which the fusion protein comprises a programmable DNAmodification protein having nuclease activity, the fusion protein cancleave one or both strands of the targeted chromosomal sequence.Double-stranded breaks can be repaired by a non-homologous end-joining(NHEJ) repair process. Because NHEJ is error-prone, indels (i.e.,deletions or insertions) of at least one base pair, substitutions of atleast one base pair, or combinations thereof can occur during the repairof the break. Accordingly, the targeted chromosomal sequence can bemodified, mutated, or inactivated. For example, a deletion, insertion,or substitution in the reading frame of a coding sequence can lead to analtered protein product, or no protein product (which is termed a “knockout”). In some iterations, the method can further comprise introducinginto the cell a donor polynucleotide (see below) comprising a donorsequence that is flanked by sequence having substantial sequenceidentity to sequences located on either side of the target chromosomalsequence, such that during repair of the double-stranded break by ahomology directed repair process (HDR) the donor sequence in the donorpolynucleotide can be exchanged with or integrated into the chromosomalsequence at the target chromosomal sequence. Integration of an exogenoussequence is termed a “knock in.”

In various iterations, therefore, the efficiency of targeted genomemodification can be increased by at least about 0.1-fold, at least about0.5-fold, at least about 1-fold, at least about 2-fold, at least about5-fold, at least about 10-fold, or at least about 20-fold, at leastabout 50-fold, at least about 100-fold, or more than about 100-foldrelative to the parental programmable DNA modification protein that isnot linked to at least one nucleosome interacting protein domain.

In embodiments in which the fusion protein comprises a programmable DNAmodification protein having non-nuclease activity, the fusion proteincan modify DNA or associated proteins at the target chromosomal sequenceor modify expression of the target chromosomal sequence. For example,when the programmable DNA modification protein comprises epigeneticmodification activity, the status of histone acetylation, methylation,phosphorylation, adenylation, etc. can be modified or the status of DNAmethylation, amination, etc. can be modified. As an example, inembodiments in which the programmable DNA modification protein comprisescytidine deaminase activity, one or more cytidine residues at the targetchromosomal sequence can be converted to uridine residues.Alternatively, when the programmable DNA modification protein comprisestranscriptional activation or repressor activity, transcription attarget chromosomal sequence can be increased or decreased.

The resultant epigenetic modification or transcriptional regulation canbe increased by at least about 0.1-fold, at least about 0.5-fold, atleast about 1-fold, at least about 2-fold, at least about 5-fold, atleast about 10-fold, or at least about 20-fold, at least about 50-fold,at least about 100-fold, or more than about 100-fold relative to theparental programmable DNA modification protein that is not linked to atleast one nucleosome interacting protein domain.

The targeted genome, transcriptional, epigenetic modifications detailedabove can be performed singly or multiplexed (i.e., two or morechromosomal sequences can be targeted simultaneously).

(c) Optional Donor Polynucleotide

In embodiments in which the fusion protein comprises a programmable DNAmodification protein having nuclease activity, the method can furthercomprise introducing at least one donor polynucleotide into the cell.The donor polynucleotide can be single-stranded or double-stranded,linear or circular, and/or RNA or DNA. In some embodiments, the donorpolynucleotide can be a vector, e.g., a plasmid vector.

The donor polynucleotide comprises at least one donor sequence. In someaspects, the donor sequence of the donor polynucleotide can be amodified version of an endogenous or native chromosomal sequence. Forexample, the donor sequence can be essentially identical to a portion ofthe chromosomal sequence at or near the sequence targeted by the DNAmodification protein, but which comprises at least one nucleotidechange. Thus, upon integration or exchange with the native sequence, thesequence at the targeted chromosomal location comprises at least onenucleotide change. For example, the change can be an insertion of one ormore nucleotides, a deletion of one or more nucleotides, a substitutionof one or more nucleotides, or combinations thereof. As a consequence ofthe “gene correction” integration of the modified sequence, the cell canproduce a modified gene product from the targeted chromosomal sequence.

In other aspects, the donor sequence of the donor polynucleotide can bean exogenous sequence. As used herein, an “exogenous” sequence refers toa sequence that is not native to the cell, or a sequence whose nativelocation is in a different location in the genome of the cell. Forexample, the exogenous sequence can comprise protein coding sequence,which can be operably linked to an exogenous promoter control sequencesuch that, upon integration into the genome, the cell is able to expressthe protein coded by the integrated sequence. Alternatively, theexogenous sequence can be integrated into the chromosomal sequence suchthat its expression is regulated by an endogenous promoter controlsequence. In other iterations, the exogenous sequence can be atranscriptional control sequence, another expression control sequence,an RNA coding sequence, and so forth. As noted above, integration of anexogenous sequence into a chromosomal sequence is termed a “knock in.”

As can be appreciated by those skilled in the art, the length of thedonor sequence can and will vary. For example, the donor sequence canvary in length from several nucleotides to hundreds of nucleotides tohundreds of thousands of nucleotides.

Typically, the donor sequence in the donor polynucleotide is flanked byan upstream sequence and a downstream sequence, which have substantialsequence identity to sequences located upstream and downstream,respectively, of the sequence targeted by the programmable DNAmodification protein. Because of these sequence similarities, theupstream and downstream sequences of the donor polynucleotide permithomologous recombination between the donor polynucleotide and thetargeted chromosomal sequence such that the donor sequence can beintegrated into (or exchanged with) the chromosomal sequence.

The upstream sequence, as used herein, refers to a nucleic acid sequencethat shares substantial sequence identity with a chromosomal sequenceupstream of the sequence targeted by the programmable DNA modificationprotein. Similarly, the downstream sequence refers to a nucleic acidsequence that shares substantial sequence identity with a chromosomalsequence downstream of the sequence targeted by the programmable DNAmodification protein. As used herein, the phrase “substantial sequenceidentity” refers to sequences having at least about 75% sequenceidentity. Thus, the upstream and downstream sequences in the donorpolynucleotide can have about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity with sequence upstream or downstreamto the target sequence. In an exemplary embodiment, the upstream anddownstream sequences in the donor polynucleotide can have about 95% or100% sequence identity with chromosomal sequences upstream or downstreamto the sequence targeted by the programmable DNA modification protein.

In some embodiments, the upstream sequence shares substantial sequenceidentity with a chromosomal sequence located immediately upstream of thesequence targeted by the programmable DNA modification protein. In otherembodiments, the upstream sequence shares substantial sequence identitywith a chromosomal sequence that is located within about one hundred(100) nucleotides upstream from the target sequence. Thus, for example,the upstream sequence can share substantial sequence identity with achromosomal sequence that is located about 1 to about 20, about 21 toabout 40, about 41 to about 60, about 61 to about 80, or about 81 toabout 100 nucleotides upstream from the target sequence. In someembodiments, the downstream sequence shares substantial sequenceidentity with a chromosomal sequence located immediately downstream ofthe sequence targeted by the programmable DNA modification protein. Inother embodiments, the downstream sequence shares substantial sequenceidentity with a chromosomal sequence that is located within about onehundred (100) nucleotides downstream from the target sequence. Thus, forexample, the downstream sequence can share substantial sequence identitywith a chromosomal sequence that is located about 1 to about 20, about21 to about 40, about 41 to about 60, about 61 to about 80, or about 81to about 100 nucleotides downstream from the target sequence.

Each upstream or downstream sequence can range in length from about 20nucleotides to about 5000 nucleotides. In some embodiments, upstream anddownstream sequences can comprise about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200,3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 nucleotides. Inspecific embodiments, upstream and downstream sequences can range inlength from about 50 to about 1500 nucleotides.

(d) Cell Types

A variety of cells are suitable for use in the methods disclosed herein.In general, the cell is a eukaryotic cell. For example, the cell can bea human cell, a non-human mammalian cell, a non-mammalian vertebratecell, an invertebrate cell, an insect cell, a plant cell, a yeast cell,or a single cell eukaryotic organism. In some embodiments, the cell canalso be a one cell embryo. For example, a non-human mammalian embryoincluding rat, hamster, rodent, rabbit, feline, canine, ovine, porcine,bovine, equine, and primate embryos. In still other embodiments, thecell can be a stem cell such as embryonic stem cells, ES-like stemcells, fetal stem cells, adult stem cells, and the like. In oneembodiment, the stem cell is not a human embryonic stem cell.Furthermore, the stem cells may include those made by the techniquesdisclosed in WO2003/046141, which is incorporated herein in itsentirety, or Chung et al. (Cell Stem Cell, 2008, 2:113-117). The cellcan be in vitro or in vivo (i.e., within an organism). In exemplaryembodiments, the cell is a mammalian cell or mammalian cell line. Inparticular embodiments, the cell is a human cell or human cell line.

Non-limiting examples of suitable mammalian cells or cell lines includehuman embryonic kidney cells (HEK293, HEK293T); human cervical carcinomacells (HELA); human lung cells (W138); human liver cells (Hep G2); humanU2-OS osteosarcoma cells, human A549 cells, human A-431 cells, and humanK562 cells; Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK)cells; mouse myeloma NSO cells, mouse embryonic fibroblast 3T3 cells(NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mousemyoblast C2C12 cells; mouse myeloma SP2/0 cells; mouse embryonicmesenchymal C3H-10T1/2 cells; mouse carcinoma CT26 cells, mouse prostateDuCuP cells; mouse breast EMT6 cells; mouse hepatoma Hepalclc7 cells;mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mousemyocardial MyEnd cells; mouse renal RenCa cells; mouse pancreatic RIN-5Fcells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; ratglioblastoma 9 L cells; rat B lymphoma RBL cells; rat neuroblastoma B35cells; rat hepatoma cells (HTC); buffalo rat liver BRL 3A cells; caninekidney cells (MDCK); canine mammary (CMT) cells; rat osteosarcoma D17cells; rat monocyte/macrophage DH82 cells; monkey kidney SV-40transformed fibroblast (COS7) cells; monkey kidney CVI-76 cells; Africangreen monkey kidney (VERO-76) cells. An extensive list of mammalian celllines may be found in the American Type Culture Collection catalog(ATCC, Manassas, Va.).

(VII) Methods for Detecting Specific Genomic Loci

In embodiments in which the fusion protein comprises a programmable DNAmodification having non-nuclease activity or the CRISPR complexcomprises a catalytically inactive CRISPR protein having non-nucleaseactivity, said fusion protein or CRISPR complex can be used in methodsfor detecting or visualizing specific genomic loci in eukaryotic cells.In such embodiments, the fusion protein or CRISPR protein of the complexfurther comprises at least one detectable label, such as a fluorophore(e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halotags, or suitable fluorescent dye), a detection tag (e.g., biotin,digoxigenin, and the like), quantum dots, or gold particles.Alternatively, the guide RNA of the CRISPR complex can further comprisea detectable label for in situ detection (e.g., FISH or CISH). The atleast one nucleosome interacting protein domain of the fusion protein orCRISPR complex increases access of the programmable DNA modificationprotein or CRISPR protein having non-nuclease activity to the targetchromosomal sequence, thereby enhancing detection of specific genomicloci or targeted chromosomal sequences.

The method comprises introducing into the eukaryotic cell the detectablylabeled fusion protein, detectably labeled CRISPR complex, or encodingnucleic acid, and detecting the labeled programmable DNA modificationprotein or labeled CRISPR protein bound to the target chromosomalsequence. The detecting can be via dynamic live cell imaging,fluorescent microscopy, confocal microscopy, immunofluorescence,immunodetection, RNA-protein binding, protein-protein binding, and thelike. The detecting step can be performed in live cells or fixed cells.

In embodiments in which the method comprises detecting chromatinstructural dynamics in live cells, the detectably labeled fusion proteinor detectably labeled CRISPR complex can be introduced into the cell asproteins or nucleic acids. In embodiments in which the method comprisesdetecting the targeted chromosomal sequence in fixed cells, thedetectably labeled fusion protein or detectably labeled CRISPR complexcan be introduced into the cell as proteins (or protein-RNA complexes).Means for fixing and permeabilizing cells are well known in the art. Insome embodiments, the fixed cells can be subjected to chemical and/orthermal denaturation processes to convert double-stranded chromosomalDNA into single-stranded DNA. In other embodiments, the fixed cells arenot subjected to chemical and/or thermal denaturation processes.

(VIII) Applications

The compositions and methods disclosed herein can be used in a varietyof therapeutic, diagnostic, industrial, and research applications. Insome embodiments, the present disclosure can be used to modify anychromosomal sequence of interest in a cell, animal, or plant in order tomodel and/or study the function of genes, study genetic or epigeneticconditions of interest, or study biochemical pathways involved invarious diseases or disorders. For example, transgenic organisms can becreated that model diseases or disorders, wherein the expression of oneor more nucleic acid sequences associated with a disease or disorder isaltered. The disease model can be used to study the effects of mutationson the organism, study the development and/or progression of thedisease, study the effect of a pharmaceutically active compound on thedisease, and/or assess the efficacy of a potential gene therapystrategy.

In other embodiments, the compositions and methods can be used toperform efficient and cost effective functional genomic screens, whichcan be used to study the function of genes involved in a particularbiological process and how any alteration in gene expression can affectthe biological process, or to perform saturating or deep scanningmutagenesis of genomic loci in conjunction with a cellular phenotype.Saturating or deep scanning mutagenesis can be used to determinecritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease, for example.

In further embodiments, the compositions and methods disclosed hereincan be used for diagnostic tests to establish the presence of a diseaseor disorder and/or for use in determining treatment options. Examples ofsuitable diagnostic tests include detection of specific mutations incancer cells (e.g., specific mutation in EGFR, HER2, and the like),detection of specific mutations associated with particular diseases(e.g., trinucleotide repeats, mutations in P3-globin associated withsickle cell disease, specific SNPs, etc.), detection of hepatitis,detection of viruses (e.g., Zika), and so forth.

In additional embodiments, the compositions and methods disclosed hereincan be used to correct genetic mutations associated with a particulardisease or disorder such as, e.g., correct globin gene mutationsassociated with sickle cell disease or thalassemia, correct mutations inthe adenosine deaminase gene associated with severe combined immunedeficiency (SCID), reduce the expression of HTT, the disease-causinggene of Huntington's disease, or correct mutations in the rhodopsin genefor the treatment of retinitis pigmentosa. Such modifications may bemade in cells ex vivo.

In still other embodiments, the compositions and methods disclosedherein can be used to generate crop plants with improved traits orincreased resistance to environmental stresses. The present disclosurecan also be used to generate farm animal with improved traits orproduction animals. For example, pigs have many features that make themattractive as biomedical models, especially in regenerative medicine orxenotransplantation.

(IX) Enumerated Embodiments

The following enumerated embodiments are presented to illustrate certainaspects of the present invention, and are not intended to limit itsscope.

1. A fusion protein comprising at least one nucleosome interactingprotein domain linked to a programmable DNA modification protein.

2. The fusion protein of embodiment 1, wherein the at least onenucleosome interacting protein domain is a DNA binding domain from ahigh mobility group (HMG) box (HMGB) protein chosen from HMGB1, HMGB2,or HMGB3; a HMG nucleosome-binding (HMGN) protein chosen from HMGN1,HMGN2, HMGN3a, HMGN3b, HMGN4, or HMGN5; a central globular domain from ahistone H1 variant; a DNA binding domain from a chromatin remodelingcomplex protein chosen from switch/sucrose non-fermentable (SWI/SNF)complex, imitation switch (ISWI) complex, chromodomain-helicase-DNAbinding (CHD) complex, nucleosome remodeling and deacetylase (NuRD)complex, IN080 complex, SWR1 complex, RSC complex, or combinationthereof.

3. The fusion protein of embodiment 2, wherein the at least onenucleosome interacting protein domain is HMGB1 box A domain, HMGN1protein, HMGN2 protein, HMGN3a protein, HMGN3b protein, histone H1central globular domain, ISWI protein DNA binding domain, CHD1 proteinDNA binding domain, or combination thereof.

4. The fusion protein of any one of embodiments 1 to 3, wherein theprogrammable DNA modification protein has nuclease activity.

5. The fusion protein of embodiment 4, wherein the programmable DNAmodification protein is a clustered regularly interspersed shortpalindromic repeats (CRISPR) nuclease or nickase, a zinc finger nuclease(ZFN), a transcription activator-like effector nuclease (TALEN), ameganuclease, or a chimeric protein comprising a programmable DNAbinding domain linked to a nuclease domain.

6. The fusion protein of any one of embodiments 1 to 3, wherein theprogrammable DNA modification protein has non-nuclease activity.

7. The fusion protein of embodiment 6, wherein the programmable DNAmodification protein is a chimeric protein comprising a programmable DNAbinding domain linked to a non-nuclease domain.

8. The fusion protein of embodiment 7, wherein the programmable DNAbinding domain is a CRISPR protein modified to lack all nucleaseactivity, a zinc finger protein, or a transcription activator-likeeffector.

9. The fusion protein of embodiment 7, wherein the non-nuclease domainhas acetyltransferase activity, deacetylase activity, methyltransferaseactivity, demethylase activity, kinase activity, phosphatase activity,ubiquitin ligase activity, deubiquitinating activity, adenylationactivity, deadenylation activity, SUMOylating activity, deSUMOylatingactivity, ribosylation activity, deribosylation activity, myristoylationactivity, demyristoylation activity, citrullination activity, helicaseactivity, amination activity, deamination activity, alkylation activity,dealkylation activity, oxidation activity, transcriptional activationactivity, or transcriptional repressor activity.

10. The fusion protein of embodiment 9, wherein the non-nuclease domainhas cytosine deaminase activity, histone acetyltransferase activity,transcriptional activation activity, or transcriptional repressoractivity.

11. The fusion protein of any one of embodiments 1 to 10, wherein the atleast one nucleosome interacting protein domain is linked to theprogrammable DNA modification protein directly via a chemical bond,indirectly via a linker, or combination thereof.

12. The fusion protein of any one of embodiments 1 to 11, wherein the atleast one nucleosome interacting protein domain is linked to theprogrammable DNA modification protein at its N-terminus, C-terminus, aninternal location, or combination thereof.

13. The fusion protein of any one of embodiments 1 to 12, furthercomprising at least one nuclear localization signal, at least onecell-penetrating domain, at least one marker domain, or combinationthereof.

14. A fusion protein comprising a clustered regularly interspersed shortpalindromic repeats (CRISPR) protein linked to at least one nucleosomeinteracting protein domain.

15. The fusion protein of embodiment 14, wherein the CRISPR protein is atype II CRISPR/Cas9 nuclease or nickase, or the CRISPR protein is a typeV CRISPR/Cpf1 nuclease or nickase.

16. The fusion protein of embodiment 14, wherein the CRISPR protein is atype II CRISPR/Cas9 protein modified to lack all nuclease activity andlinked to a non-nuclease domain, or a type V CRISPR/Cpf1 proteinmodified to lack all nuclease activity and linked to a non-nucleasedomain.

17. The fusion protein of embodiment 16, wherein the non-nuclease domainhas cytosine deaminase activity, histone acetyltransferase activity,transcriptional activation activity, or transcriptional repressoractivity.

18. The fusion protein of any one of embodiments 14 to 17, wherein theat least one nucleosome interacting protein domain is a high mobilitygroup (HMG) box (HMGB) DNA binding domain, a HMG nucleosome-binding(HMGN) protein, a central globular domain from a histone H1 variant, aDNA binding domain from a chromatin remodeling complex protein, or acombination thereof.

19. The fusion protein of embodiment 18, wherein at least one nucleosomeinteracting protein domain is HMGB1 box A domain, HMGN1 protein, HMGN2protein, HMGN3a protein, HMGN3b protein, histone H1 central globulardomain, imitation switch (ISWI) protein DNA binding domain,chromodomain-helicase-DNA protein 1 (CHD1) DNA binding domain, or acombination thereof.

20. The fusion protein of any one of embodiments 14 to 19, wherein theat least one nucleosome interacting protein domain is linked to theCRISPR protein directly via a chemical bond, indirectly via a linker, ora combination thereof.

21. The fusion protein of any one of embodiments 14 to 20, wherein theat least one nucleosome interacting protein domain is linked to theCRISPR protein at its N-terminus, C-terminus, an internal location, or acombination thereof.

22. The fusion protein of any one of embodiments 14 to 21, furthercomprising at least one nuclear localization signal, at least onecell-penetrating domain, at least one marker domain, or a combinationthereof.

23. The fusion protein of any one of embodiments 14 to 22, wherein theCRISPR protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcusthermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9),Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9),Francisella novicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9),Neisseria meningitis Cas9 (NmCas9), Francisella novicida Cpf1 (FnCpf1),Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006Cpf1 (LbCpf1).

24. The fusion protein of any one of embodiments 14 to 23, wherein thefusion protein has an amino acid sequence having at least about 90%sequence identity with SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, or SEQ IDNO:79.

25. The fusion protein of any one of embodiments 14 to 24, wherein thefusion protein has an amino acid sequence as set forth in SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79.

26. A complex comprising at least one fusion protein of any one ofembodiments 14 to 25 and at least one guide RNA.

27. A nucleic acid encoding the fusion protein of any one of embodiments1 to 25.

28. The nucleic acid of embodiment 27, which is codon optimized fortranslation in a eukaryotic cell.

29. The nucleic acid of embodiments 27 or 28, which is part of a viralvector, a plasmid vector, or a self-replicating RNA.

30. A method for increasing efficiency of targeted genome or epigeneticmodification in a eukaryotic cell, the method comprising introducinginto the eukaryotic cell at least one fusion protein as set forth in anyone of embodiments 1 to 25, or nucleic acid encoding the at least onefusion protein as set forth in any one of embodiments 27 to 29, whereinthe programmable DNA modification protein of the at least one fusionprotein is targeted to a target chromosomal sequence and the at leastone nucleosome interacting protein domain of the at least one fusionprotein alters nucleosomal or chromatin structure such that the at leastone fusion protein has increased access to the target chromosomalsequence, thereby increasing efficiency of targeted genome or epigeneticmodification.

31. The method of embodiment 30, wherein the DNA modification protein ofthe at least one fusion protein comprises a CRISPR protein and themethod further comprises introducing into the eukaryotic cell at leastone guide RNA or nucleic acid encoding the at least one guide RNA.

32. The method of embodiments 30 or 31, wherein the method furthercomprises introducing into the eukaryotic cell at least one donorpolynucleotide, the donor polynucleotide comprising at least one donorsequence.

33. The method of any one of embodiments 30 to 32, wherein theeukaryotic cell is in vitro.

34. The method of any one of embodiments 30 to 32, wherein theeukaryotic cell is in vivo.

35. The method of any one of embodiments 30 to 34, wherein theeukaryotic cell is a mammalian cell.

36. The method of any one of embodiments 30 to 35, wherein theeukaryotic cell is a human cell.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd Ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

When introducing elements of the present disclosure or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “about” when used in relation to a numerical value, x, forexample means x±5%.

As used herein, the terms “complementary” or “complementarity” refer tothe association of double-stranded nucleic acids by base pairing throughspecific hydrogen bonds. The base paring may be standard Watson-Crickbase pairing (e.g., 5′-A G T C-3′ pairs with the complementary sequence3′-T C A G-5′). The base pairing also may be Hoogsteen or reversedHoogsteen hydrogen bonding. Complementarity is typically measured withrespect to a duplex region and thus, excludes overhangs, for example.Complementarity between two strands of the duplex region may be partialand expressed as a percentage (e.g., 70%), if only some (e.g., 70%) ofthe bases are complementary. The bases that are not complementary are“mismatched.” Complementarity may also be complete (i.e., 100%), if allthe bases in the duplex region are complementary.

As used herein, the term “CRISPR system” refers to a complex comprisinga CRISPR protein (i.e., nuclease, nickase, or catalytically deadprotein) and a guide RNA.

The term “endogenous sequence,” as used herein, refers to a chromosomalsequence that is native to the cell.

As used herein, the term “exogenous” refers to a sequence that is notnative to the cell, or a chromosomal sequence whose native location inthe genome of the cell is in a different chromosomal location.

A “gene,” as used herein, refers to a DNA region (including exons andintrons) encoding a gene product, as well as all DNA regions whichregulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites, and locus control regions.

The term “heterologous” refers to an entity that is not endogenous ornative to the cell of interest. For example, a heterologous proteinrefers to a protein that is derived from or was originally derived froman exogenous source, such as an exogenously introduced nucleic acidsequence. In some instances, the heterologous protein is not normallyproduced by the cell of interest.

The term “nickase” refers to an enzyme that cleaves one strand of adouble-stranded nucleic acid sequence (i.e., nicks a double-strandedsequence). For example, a nuclease with double strand cleavage activitycan be modified by mutation and/or deletion to function as a nickase andcleave only one strand of a double-stranded sequence.

The term “nuclease,” as used herein, refers to an enzyme that cleavesboth strands of a double-stranded nucleic acid sequence.

The terms “nucleic acid” and “polynucleotide” refer to adeoxyribonucleotide or ribonucleotide polymer, in linear or circularconformation, and in either single- or double-stranded form. For thepurposes of the present disclosure, these terms are not to be construedas limiting with respect to the length of a polymer. The terms canencompass known analogs of natural nucleotides, as well as nucleotidesthat are modified in the base, sugar and/or phosphate moieties (e.g.,phosphorothioate backbones). In general, an analog of a particularnucleotide has the same base-pairing specificity; i.e., an analog of Awill base-pair with T.

The term “nucleotide” refers to deoxyribonucleotides or ribonucleotides.The nucleotides may be standard nucleotides (i.e., adenosine, guanosine,cytidine, thymidine, and uridine), nucleotide isomers, or nucleotideanalogs. A nucleotide analog refers to a nucleotide having a modifiedpurine or pyrimidine base or a modified ribose moiety. A nucleotideanalog may be a naturally occurring nucleotide (e.g., inosine,pseudouridine, etc.) or a non-naturally occurring nucleotide.Non-limiting examples of modifications on the sugar or base moieties ofa nucleotide include the addition (or removal) of acetyl groups, aminogroups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methylgroups, phosphoryl groups, and thiol groups, as well as the substitutionof the carbon and nitrogen atoms of the bases with other atoms (e.g.,7-deaza purines). Nucleotide analogs also include dideoxy nucleotides,2′-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleicacids (PNA), and morpholinos.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues.

As used herein, the term “programmable DNA modification protein” refersto a protein that is engineered to bind a specific target sequence inchromosomal DNA and which modifies the DNA or protein(s) associated withDNA at or near the target sequence.

The term “sequence identity” as used herein, indicates a quantitativemeasure of the degree of identity between two sequences of substantiallyequal length. The percent identity of two sequences, whether nucleicacid or amino acid sequences, is the number of exact matches between twoaligned sequences divided by the length of the shorter sequence andmultiplied by 100. An approximate alignment for nucleic acid sequencesis provided by the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981). This algorithm can beapplied to amino acid sequences by using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An exemplary implementation of this algorithm to determinepercent identity of a sequence is provided by the Genetics ComputerGroup (Madison, Wis.) in the “BestFit” utility application. Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art, for example, anotheralignment program is BLAST, used with default parameters. For example,BLASTN and BLASTP can be used using the following default parameters:genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found on the GenBank website. In general, the substitutions areconservative amino acid substitutions: limited to exchanges withinmembers of group 1: glycine, alanine, valine, leucine, and Isoleucine;group 2: serine, cysteine, threonine, and methionine; group 3: proline;group 4: phenylalanine, tyrosine, and tryptophan; group 5: aspartate,glutamate, asparagine, and glutamine.

The terms “target sequence,” “target chromosomal sequence,” and “targetsite” are used interchangeably to refer to the specific sequence inchromosomal DNA to which the programmable DNA modification protein istargeted, and the site at which the programmable DNA modificationprotein modifies the DNA or protein(s) associated with the DNA.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion.

In general, identity refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Two or more sequences(polynucleotide or amino acid) can be compared by determining theirpercent identity.

As various changes could be made in the above-described cells andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

EXAMPLES

The following examples illustrate certain aspects of the disclosure.Table 1 lists the peptide sequences of nucleosome interacting domainsand Table 2 presents target chromosomal sequences used in Examples 1-8presented below.

TABLE 1 Peptide Sequences of Nucleosome Interacting Domains NucleosomeInteracting SEQ Domain Sequence (NH₂—COOH) ID NO: Human HMGB1MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNF 40 box A domainSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMK (1-84 aa) TYIPPKGE Human HMGN1MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKA 41 proteinAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAE NGETKTEESPASDEAGEKEAKSDHuman HMGN2 MPKRKAEGDAKGDKAKVKDEPQRRSARLSAKPAPPKPE 42 proteinPKPKKAPAKKGEKVPKGKKGKADAGKEGNNPAENGDAK TDQAQKAEGAGDAK HumanMPKRKSPENTEGKDGSKVTKQEPTRRSARLSAKPAPPK 43 HMGN3aPEPKPRKTSAKKEPGAKISRGAKGKKEEKQEAGKEGTAP protein SENGETKAEEAQKTESVDNEGEHuman MPKRKSPENTEGKDGSKVTKQEPTRRSARLSAKPAPPK 44 HMGN3bPEPKPRKTSAKKEPGAKISRGAKGKKEEKQEAGKEGTEN protein Human histoneSTDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVG 45 H1 centralENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEP globular domain (22-101 aa)Yeast ISWI LLNPTKRERKENYSIDNYYKDVLNTGRSSTPSHPRMPKP 46 chromatin-HVFHSHQLQPPQLKVLYEKERMVVTAKKTGYVPTMDDVK remodelingAAYGDISDEEEKKQKLELLKLSVNNSQPLTEEEEKMKAD complexWESEGFTNWNKLEFRKFITVSGKYGRNSIQAIARELAPGK ATPase ISW1TLEEVRAYAKAFWSNIERIEDYEKYLKIIENEEEKIKRVKM DNA bindingQQEALRRKLSEYKNPFFDLKLKHPPSSNNKRTYSEEEDR domainFILLMLFKYGLDRDDVYELVRDEIRDCPLFELDFYFRSRTPVELARRGNTLLQCLEKEFNAGIVLDDATKDRMKKEDENGKRIREEFADQTANEKENVDGVESKKAKIEDTSNVGTEQLV AEKIPENETTH Yeast chromoDMDSIGESEVRALYKAILKFGNLKEILDELIADGTLPVKSFE 47 domain-KYGETYDEMMEAAKDCVHEEEKNRKEILEKLEKHATAYR containingAKLKSGEIKAENQPKDNPLTRLSLKKREKKAVLFNFKGVK protein 1SLNAESLLSRVEDLKYLKNLINSNYKDDPLKFSLGNNTPK (CHD1) DNAPVQNWSSNVVTKEEDEKLLIGVFKYGYGSVVTQIRDDPFL binding domainGITDKIFLNEVHNPVAKKSASSSDTTPTPSKKGKGITGSSK KVPGAIHLGRRVDYLLSFLRGGLNTKSPS

TABLE 2 Chromosomal Target Sites Locus Site Sequence (5′-3′) SEQ ID NO:Streptococcus pyogenes Cas9 (SpCas9) POR #1 AGCCGTGAGTGGAGGGAGCGTGG 48POR #2 AGAGGGAGGGGTTGGACTACAGG 49 POR #3 CATTCGCCAGTACGAGCTTGTGG 50 CAR#1 CTTTAATGCGCTGACTTGTGAGG 51 EMX1 #1 GTGGCGCATTGCCACGAAGCAGG 52 EMX1 #2TTCTTCTTCTGCTCGGACTCAGG 53 Streptococcus pasteurianus Cas9 (SpaCas9) POR#1 TGCTGGAAAGGGGAGACCAAGGGTGA 54 POR #2 AGAGCTACGAGAACCAGAAGCCGTGA 55Francisella novicida Cpf1 (FnCpf1) POR #1 TTCCCGGCCTCACCCTTGGTCTCCCC 56POR #2 TTGGTCTCCCCTTTCCAGCATTCGCC 57 POR #3 TTCCAGCATTCGCCAGTACGAGCTTG58 Campylobacter jejuni Cas9 (CjCas9) POR #1GATCAACATGGGAGACTCCCACGTGGACAC 59 POR #2 AGATACTTCTTCGGCCACCGCCTCGGACAC60

Example 1. Improvement of Streptococcus pyogenes Cas9 (SpCas9) ActivityUsing Human HMGB1 Box a Domain

A human HMGB1 box A domain (SEQ ID NO:40) was fused with SpCas9 (+NLS)at the nuclease carboxyl terminus with the linker LEGGGS (SEQ ID NO:1)between Cas9 and the HMGB1 box A domain. Human K562 cells (1×10⁶) weretransfected with plasmid DNA encoding the fusion protein or wild typeSpCas9 protein in molar equivalent amounts (5.2 and 5.0 μg for thefusion protein and the wild type Cas9 protein, respectively) incombination with 3 μg of a sgRNA plasmid for targeting a genomic site(#1) in the human cytochrome p450 oxidoreductase (POR) locus.Transfection was carried out using nucleofection on an Amaxinucleofector. Three days after transfection, cells were lysed with a DNAextraction solution (QuickExtract™) and the targeted genomic region wasPCR amplified. Cas9 nuclease target cleavage activities (% indels) weremeasured using Cel-I assays. As shown in Table 3, fusion of the humanHMGB1 box A domain with the nuclease increased SpCas9 cleavageefficiency at the target site.

TABLE 3 Cleavage Efficiency Nuclease Target Site Indel (%) Wild typeSpCas9 POR/site #1 8.5 SpCas9-HMGB1 box A fusion POR/site #1 21.3

Example 2. Improvement of Streptococcus pyogenes Cas9 (SpCas9) ActivityUsing Human HMGN1, HMGN2, HMGN3a, and HMGN3b

Human HMGN1, HMGN2, HMGN3a, and HMGN3b (SEQ ID NOS:41-44, respectively)were each fused with SpCas9 (+NLS) at the nuclease carboxyl terminuswith the linker LEGGGS (SEQ ID NO:1) between Cas9 and each of the HMGNpeptides. Human K562 cells (1×10⁶) were transfected with plasmid DNAencoding each of the fusion proteins or the wild type SpCas9 protein inmolar equivalent amounts (5.2 and 5.0 μg for each of the fusion proteinsand the wild type Cas9 protein, respectively) in combination with 3 μgof a sgRNA plasmid for targeting a genomic site (#1) in the humancytochrome p450 oxidoreductase (POR) locus. Transfection was carried outusing nucleofection on an Amaxi nucleofector. Three days aftertransfection, cells were lysed with a DNA extraction solution(QuickExtract™) and the targeted genomic region was PCR amplified. Cas9target cleavage activities (% indels) were measured using Cel-I assays.The results, as summarized in Table 4, show that fusion of each of thehuman HMGN peptides with the nuclease increased SpCas9 cleavageefficiency at the target site.

TABLE 4 Cleavage Efficiency Nuclease Target Site Indel (%) Wild typeSpCas9 POR/site #1 8.5 SpCas9-HMGN1 fusion POR/site #1 18.3 SpCas9-HMGN2fusion POR/site #1 13.3 SpCas9-HMGN3a fusion POR/site #1 13.5SpCas9-HMGN3b fusion POR/site #1 14.4

Example 3. Improvement of Streptococcus pyogenes Cas9 (SpCas9) ActivityUsing Human Histone H1 Central Globular Domain

A human histone H1 central globular domain (SEQ ID NO:45) was fused withSpCas9 (+NLS) at the nuclease carboxyl terminus with the linker LEGGGS(SEQ ID NO:1) between Cas9 and the globular domain. Human K562 cells(1×10⁶) were transfected with plasmid DNA encoding the fusion protein orthe wild type SpCas9 protein in molar equivalent amounts (5.2 and 5.0 μgfor the fusion protein and the wild type Cas9 protein, respectively) incombination with 3 μg of a sgRNA plasmid for targeting a genomic site(#1) in the human cytochrome p450 oxidoreductase (POR) locus.Transfection was carried out using nucleofection on an Amaxinucleofector. Three days after transfection, cells were lysed with a DNAextraction solution (QuickExtract™) and the targeted genomic region wasPCR amplified. Cas9 target cleavage activities (% indels) were measuredusing Cel-I assays. The results are presented in Table 5. Fusion of thehuman histone H1 central globular domain with the nuclease increasedSpCas9 cleavage efficiency at the target site.

TABLE 5 Cleavage Efficiency Nuclease Target Site Indel (%) Wild typeSpCas9 POR/site #1 8.5 SpCas9-H1 central globular POR/site #1 19.4domain fusion

Example 4. Improvement of Streptococcus pyogenes Cas9 (SpCas9) ActivityUsing a Chromatin Remodeling Protein DNA Binding Domain

SpCas9 (+NLS) was fused with the DNA binding domain of the yeast ISWIchromatin-remodeling complex ATPase ISW1 (SEQ ID NO:46) at the nucleaseamino terminus with the linker TGSG (SEQ ID NO:2) between Cas9 and theDNA binding domain. Independently, the wild type SpCas9 was fused withthe DNA binding domain of the yeast chromo domain-containing protein 1(CHD1) (SEQ ID NO:47) at the nuclease carboxyl terminus with the linkerLEGGGS (SEQ ID NO:1) between Cas9 and the DNA binding domain. Human K562cells (1×10⁶) were transfected with plasmid DNA encoding each of thefusion proteins or the wild type SpCas9 protein in molar equivalentamounts (6.0 and 5.0 μg for each of the fusion proteins and the wildtype Cas9 protein, respectively) in combination with 3 μg of a sgRNAplasmid for targeting a genomic site (#1) in the human cytochrome p450oxidoreductase (POR) locus. Transfection was carried out usingnucleofection on an Amaxi nucleofector. Three days after transfection,cells were lysed with a DNA extraction solution (QuickExtract™) and thetargeted genomic region was PCR amplified. Cas9 target cleavageactivities (% indels) were measured using Cel-I assays. The results, assummarized in Table 6, show that the fusion of each of the DNA bindingdomains with the nuclease increased SpCas9 cleavage efficiency at thetarget site.

TABLE 6 Cleavage Efficiency Nuclease Target Site Indel (%) Wild typeSpCas9 POR/site #1 8.5 ISW1 DNA binding domain- POR/site #1 21.1 SpCas9fusion SpCas9-CHD1 DNA binding POR/site #1 20.8 domain fusion

Example 5. Improvement of Streptococcus pyogenes Cas9 (SpCas9) ActivityUsing Combinations of Nucleosome Interacting Domains

SpCas9 (+NLS) was fused with the human HMGN1 (SEQ ID N:41) at thenuclease amino terminus with the linker TGSG (SEQ ID NO:2) between Cas9and HMGN1 and with the human HMGB1 box A domain (SEQ ID NO:40) or thehuman histone H1 central globular domain (SEQ ID NO: 45) or the yeastchromo domain-containing protein 1 (CHD1) DNA binding domain (SEQ IDNO:47) at the nuclease carboxyl terminus with the linker LEGGGS (SEQ IDNO:1) between Cas9 and each of the protein domains. Human K562 cells(1×10⁶) were transfected with plasmid DNA encoding each of the fusionproteins or the wild type SpCas9 protein in molar equivalent amounts(5.4 μg for the HMGB1 box A and H1 central globular domain fusionproteins, 6.0 μg for the CHD1 DNA binding domain fusion protein, and 5.0μg for the wild type Cas9 protein) in combination with 3 μg of a sgRNAplasmid for targeting a genomic site (#1, #2, #3) in the humancytochrome p450 oxidoreductase (POR) locus, or a genomic site (#1) thehuman nuclear receptor subfamily 1 group I member 3 (CAR) locus, or agenomic site (#1, #2) the human empty spiracles homeobox 1 (EMX1) locus.Transfection was carried out using nucleofection on an Amaxinucleofector. Five days after transfection, cells were lysed with a DNAextraction solution (QuickExtract™) and each targeted genomic region wasPCR amplified. Cas9 target cleavage activities (% indels) were measuredusing Cel-I assays. The results, as summarized in the Table 7, show thatthe combinatory fusion of these protein domains with the nucleaseincreased SpCas9 cleavage efficiency at the target sites.

TABLE 7 Cleavage Efficiency Nuclease Target Site Indel (%) Wild typeSpCas9 POR/site #1 3.4 POR/site #2 1.3 POR/site #3 22.2 CAR/site #1 2.1EMX1/site #1 2.2 EMX1/site #2 1.1 HMGN1-SpCas9-HMGB1 box A fusionPOR/site #1 28.2 POR/site #2 8.3 POR/site #3 42.7 CAR/site #1 14.3EMX1/site #1 29.0 EMX1/site #2 12.1 HMGN1-SpCas9-H1 central globularPOR/site #1 24.3 domain fusion POR/site #2 6.5 POR/site #3 44.2 CAR/site#1 23.9 EMX1/site #1 26.9 EMX1/site #2 21.0 HMGN1-SpCas9-CHD1 DNAbinding POR/site #1 21.5 domain fusion POR/site #2 3.6 POR/site #3 39.8CAR/site #1 9.0 EMX1/site #1 23.5 EMX1/site #2 20.2

Example 6. Improvement of Streptococcus pasteurianus Cas9 (SpaCas9)Activity Using Combinations of Nucleosome Interacting Domains

Streptococcus pasteurianus Cas9 (SpaCas9) (+NLS) was fused with thehuman HMGN1 (SEQ ID NO:41) at the nuclease amino terminus with thelinker TGSG (SEQ ID NO:2) between Cas9 and HMGN1 and with the humanHMGB1 box A domain (SEQ ID NO:41) or the human histone H1 centralglobular domain (SEQ ID NO:45) or the yeast chromo domain-containingprotein 1 (CHD1) DNA binding domain (SEQ ID NO:47) at the nucleasecarboxyl terminus with the linker LEGGGS (SEQ ID NO:1) between Cas9 andeach of the protein domains. Human K562 cells (1×10⁶) were transfectedwith plasmid DNA encoding each of the fusion proteins or the wild typeSpaCas9 protein in molar equivalent amounts (5.4 and 5.0 μg for each ofthe fusion proteins and the wild type Cas9 protein, respectively) incombination with 3 μg of a sgRNA plasmid for targeting a genomic site(#1, #2) in the human cytochrome p450 oxidoreductase (POR) locus.Transfection was carried out using nucleofection on an Amaxinucleofector. Three days after transfection, cells were lysed with a DNAextraction solution (QuickExtract™) and the targeted genomic region wasPCR amplified. Cas9 target cleavage activities (% indels) were measuredusing Cel-I assays. As summarized in Table 8, the combinatory fusion ofthese protein domains with the nuclease increased SpaCas9 cleavageefficiency at the target sites.

TABLE 8 Cleavage Efficiency Nuclease Target Site Indel (%) Wild typeSpaCas9 POR/site #1 16.6 POR/site #2 12.9 HMGN1-SpaCas9-HMGB1 box APOR/site #1 20.6 fusion POR/site #2 35.8 HMGN1-SpaCas9-H1 centralglobular POR/site #1 28.6 domain fusion POR/site #2 31.7HMGN1-SpaCas9-CHD1 DNA binding POR/site #1 19.4 domain fusion POR/site#2 18.5

Example 7. Improvement of Francisella novicida Cpf1 (FnCpf1) ActivityUsing Combinations of Nucleosome Interacting Domains

Francisella novicida Cpf1 (FnCpf1) (+NLS) was fused with the human HMGN1(SEQ ID NO:41) at the nuclease amino terminus with the linker TGSG (SEQID NO:2) between Cpf1 and HMGN1 and with the human HMGB1 box A domain(SEQ ID NO:40) or the human histone H1 central globular domain (SEQ IDNO:45) or the yeast chromo domain-containing protein 1 (CHD1) DNAbinding domain (SEQ ID NO:47) at the nuclease carboxyl terminus with thelinker LEGGGS (SEQ ID NO:1) between Cpf1 and each of the proteindomains. Human K562 cells (1×10⁶) were transfected with plasmid DNAencoding each of the fusion proteins or the wild type FnCpf1 protein inmolar equivalent amounts (5.4 and 5.0 μg for each of the fusion proteinsand the wild type Cas9 protein, respectively) in combination with 3 μgof a sgRNA plasmid for targeting a genomic site (#1, #2, #3) in thehuman cytochrome p450 oxidoreductase (POR) locus. Transfection wascarried out using nucleofection on an Amaxi nucleofector. Three daysafter transfection, cells were lysed with a DNA extraction solution(QuickExtract™) and the targeted genomic region was PCR amplified. Cas9target cleavage activities (% indels) were measured using Cel-I assays.The results, as summarized in Table 9, show that the combinatory fusionof these protein domains with the nuclease increased FnCpf1 cleavageefficiency on the target sites.

TABLE 9 Cleavage Efficiency Nuclease Target Site Indel (%) WildtypeFnCpf1 POR/site #1 2.3 POR/site #2 5.3 POR/site #3 3.0HMGN1-FnCpf1-HMGB1 box A POR/site #1 8.2 fusion POR/site #2 12.8POR/site #3 13.2 HMGN1-FnCpf1-H1 central globular POR/site #1 8.7 domainfusion POR/site #2 12.9 POR/site #3 13.2 HMGN1-FnCpf1-CHD1 DNA bindingPOR/site #1 7.7 domain fusion POR/site #2 7.5 POR/site #3 9.4

Example 8. Improvement of Campylobacter jejuni Cas9 (CjCas9) GeneEditing Efficiency

Campylobacter jejuni Cas9 (CjCas9) (+NLS) was fused with the human HMGN1(SEQ ID NO:41) at the nuclease amino terminus with the linker TGSG (SEQID NO:2) between Cas9 and HMGN1 and with the human HMGB1 box A domain(SEQ ID NO:40) or the human histone H1 central globular domain (SEQ IDNO:45) at the nuclease carboxyl terminus with the linker LEGGGS (SEQ IDNO:1) between Cas9 and each of the protein domains. The wild type CjCas9gRNA was modified by introducing a U to C mutation into the crRNAconstant repeat region and a corresponding A to G mutation into the 5′region of the tracrRNA sequence. The modified sgRNA sequence is:5′-NNNNNNNNNNNNNNNNNNNNNNGUUCUAGUCCCUGAAAAGGGACUAGAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′, where the mutatednucleotides in the crRNA and tracrRNA moieties are underlined. Guidesequences targeting two different sites (#1, #2) in the human cytochromep450 oxidoreductase gene (POR) were cloned into the wild type and themodified CjCas9 sgRNA scaffold, respectively. The expression of thesgRNAs was under the control of a U6 promoter. Human K562 cells (1×10⁶)were transfected with 4 μg of CjCas9 plasmid DNA and 3 μg of a sgRNAplasmid DNA. Transfection was carried out using nucleofection on anAmaxi nucleofector. Three days after transfection, cells were lysed withQuickExtract and the targeted genomic regions were PCR amplified. CjCas9target DNA cleavage activities (% indels) were measured using Cel-Iassays. The results are presented in FIG. 1 and show that the fusionproteins had increased cleavage efficiency on the target sites, and thatmodified CjCas9 sgRNA scaffold effectively increased CjCas9 cleavageefficiency on target sites.

Table 10 presents the amino acid sequences of the specific fusionproteins. The nucleosome interacting protein domains are shown in bold,the linkers are shown in italics, and the NLS is underlined.

TABLE 10 CRISPR Fusion Proteins SpCas9- HMGB1 box A fusion(SEQ ID NO: 61)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSMGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGESpCas9-HMGN1 fusion (SEQ ID NO: 62)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSMPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD SpCas9-HMGN2 fusion (SEQ ID NO: 63)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSMPKRKAEGDAKGDKAKVKDEPQRRSARLSAKPAPPKPEPKPKKAPAKKGEKVPKGKKGKADAGKEGNNPAENGDAKTDQAQKAEGAGDAKSpCas9-HMGN3a fusion (SEQ ID NO: 64)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSMPKRKSPENTEGKDGSKVTKQEPTRRSARLSAKPAPPKPEPKPRKTSAKKEPGAKISRGAKGKKEEKQEAGKEGTAPSENGETKAEEAQKTESVDNEGE SpCas9-HMGN3b fusion (SEQ ID NO: 65)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSMPKRKSPENTEGKDGSKVTKQEPTRRSARLSAKPAPPKPEPKPRKTSAKKEPGAKISRGAKGKKEEKQEAGKEGTENSpCas9-Histone H1 globular fusion (SEQ ID NO: 66)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSSTDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEP ISWI-SpCas9 fusion(SEQ ID NO: 67)LLNPTKRERKENYSIDNYYKDVLNTGRSSTPSHPRMPKPHVFHSHQLQPPQLKVLYEKERMWTAKKTGYVPTMDDVKAAYGDISDEEEKKQKLELLKLSVNNSQPLTEEEEKMKADWESEGFTNWNKLEFRKFITVSGKYGRNSIQAIARELAPGKTLEEVRAYAKAFWSNIERIEDYEKYLKIIENEEEKIKRVKMQQEALRRKLSEYKNPFFDLKLKHPPSSNNKRTYSEEEDRFILLMLFKYGLDRDDVYELVRDEIRDCPLFELDFYFRSRTPVELARRGNTLLQCLEKEFNAGIVLDDATKDRMKKEDENGKRIREEFADQTANEKENVDGVESKKAKIEDTSNVGTEQLVAEKIPENETTHTGSGMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKV SpCas9-CHD1 fusion (SEQ ID NO: 68)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSDMDSIGESEVRALYKAILKFGNLKEILDELIADGTLPVKSFEKYGETYDEMMEAAKDCVHEEEKNRKEILEKLEKHATAYRAKLKSGEIKAENQPKDNPLTRLSLKKREKKAVLFNFKGVKSLNAESLLSRVEDLKYLKNLINSNYKDDPLKFSLGNNTPKPVQNWSSNWTKEEDEKLLIGVFKYGYGSWTQIRDDPFLGITDKIFLNEVHNPVAKKSASSSDTTPTPSKKGKGITGSSKKVPGAIHLGRRVDYLLSFLRGGLNTKSPSHMGN1-SpCas9-HMGB1 box A fusion (SEQ ID NO: 69)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGE HMGN1-SpCas9-Histone H1 globular fusion(SEQ ID NO: 70)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSSTDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEP HMGN1-SpCas9-CDH1 fusion (SEQ ID NO: 71)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPKKKRKVLEGGGGSDMDSIGESEVRALYKAILKFGNLKEILDELIADGTLPVKSFEKYGETYDEMMEAAKDCVHEEEKNRKEILEKLEKHATAYRAKLKSGEIKAENQPKDNPLTRLSLKKREKKAVLFNFKGVKSLNAESLLSRVEDLKYLKNLINSNYKDDPLKFSLGNNTPKPVQNWSSNWTKEEDEKLLIGVFKYGYGSWTQIRDDPFLGITDKIFLNEVHNPVAKKSASSSDTTPTPSKKGKGITGSSKKVPGAIHLGRRVDYLLSFLRGGLNTKSPS HMGN1-SpaCas9-HMGB1 box A fusion(SEQ ID NO: 72)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMTNGKILGLDIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNAERRGFRGSRRLNRRKKHRVKRVRDLFEKYGIVTDFRNLNLNPYELRVKGLTEQLKNEELFAALRTISKRRGISYLDDAEDDSTGSTDYAKSIDENRRLLKNKTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNLKVSTETGKLSTEQKESLVEFAKNTATLGPAKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWHSFSAKLMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGNKQLETKIRLWYQQGERCLYSGKPISIQELVHNSNNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQSALRELGKDTKVSVVRGQFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNPMFVDYGKNQVVDKQTGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLGKIKDIYSQNGFDTFIKKYNKDKTQFLMYQKDSLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGNYHISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDIASGEQEIYRFLSRTMPNVNHYVELKPYDKEKFDNVQELVEALGEADKVGRCIKGLNKPNISIYKVRTDVLGNKYFVKKKGDKPKLDFKNNKKPKKKRKVLEGGGGSGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGEHMGN1-SpaCas9-Histone H1 globular fusion (SEQ ID NO: 73)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMTNGKILGLDIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNAERRGFRGSRRLNRRKKHRVKRVRDLFEKYGIVTDFRNLNLNPYELRVKGLTEQLKNEELFAALRTISKRRGISYLDDAEDDSTGSTDYAKSIDENRRLLKNKTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNLKVSTETGKLSTEQKESLVEFAKNTATLGPAKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWHSFSAKLMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGNKQLETKIRLWYQQGERCLYSGKPISIQELVHNSNNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQSALRELGKDTKVSVVRGQFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNPMFVDYGKNQVVDKQTGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLGKIKDIYSQNGFDTFIKKYNKDKTQFLMYQKDSLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGNYHISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDIASGEQEIYRFLSRTMPNVNHYVELKPYDKEKFDNVQELVEALGEADKVGRCIKGLNKPNISIYKVRTDVLGNKYFVKKKGDKPKLDFKNNKKPKKKRKVLEGGGGSSTDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEPHMGN1-SpaCas9-CHD1 fusion (SEQ ID NO: 74)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMTNGKILGLDIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNAERRGFRGSRRLNRRKKHRVKRVRDLFEKYGIVTDFRNLNLNPYELRVKGLTEQLKNEELFAALRTISKRRGISYLDDAEDDSTGSTDYAKSIDENRRLLKNKTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNLKVSTETGKLSTEQKESLVEFAKNTATLGPAKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWHSFSAKLMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGNKQLETKIRLWYQQGERCLYSGKPISIQELVHNSNNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQSALRELGKDTKVSVVRGQFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNPMFVDYGKNQVVDKQTGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLGKIKDIYSQNGFDTFIKKYNKDKTQFLMYQKDSLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGNYHISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDIASGEQEIYRFLSRTMPNVNHYVELKPYDKEKFDNVQELVEALGEADKVGRCIKGLNKPNISIYKVRTDVLGNKYFVKKKGDKPKLDFKNNKKPKKKRKVLEGGGGSDMDSIGESEVRALYKAILKFGNLKEILDELIADGTLPVKSFEKYGETYDEMMEAAKDCVHEEEKNRKEILEKLEKHATAYRAKLKSGEIKAENQPKDNPLTRLSLKKREKKAVLFNFKGVKSLNAESLLSRVEDLKYLKNLINSNYKDDPLKFSLGNNTPKPVQNWSSNWTKEEDEKLLIGVFKYGYGSWTQIRDDPFLGITDKIFLNEVHNPVAKKSASSSDTTPTPSKKGKGITGSSKKVPGAIHLGRRVDYLLSFLRGGLNTKSPS HMGN1-FnCpf1-HNGB1 fusion(SEQ ID NO: 75)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNPKKKRKVLEGGGGSGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGE HMGN1-FnCpf1-Histone H1 globular fusion(SEQ ID NO: 76)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNPKKKRKVLEGGGGSSTDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEP HMGN1-FnCpf1-CHD1 fusion (SEQ ID NO: 77)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNPKKKRKVLEGGGGSDMDSIGESEVRALYKAILKFGNLKEILDELIADGTLPVKSFEKYGETYDEMMEAAKDCVHEEEKNRKEILEKLEKHATAYRAKLKSGEIKAENQPKDNPLTRLSLKKREKKAVLFNFKGVKSLNAESLLSRVEDLKYLKNLINSNYKDDPLKFSLGNNTPKPVQNWSSNWTKEEDEKLLIGVFKYGYGSWTQIRDDPFLGITDKIFLNEVHNPVAKKSASSSDTTPTPSKKGKGITGSSKKVPGAIHLGRRVDYLLSFLRGGLNTKSPS HMGN1-CjCas9-HMGB1 box A fusion (SEQ ID NO: 78)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKKPKKKRKVLEGGGGSGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGEHMGN1-CjCas9-Histone H1 globular fusion (SEQ ID NO: 79)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKKPKKKRKVLEGGGGSSTDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEP

What is claimed is:
 1. A method for increasing efficiency of targetedgenome or epigenetic modification in a eukaryotic cell, the methodcomprising introducing into the eukaryotic cell: (a) at least one fusionprotein or nucleic acid encoding at least one fusion protein, eachfusion protein comprising a CRISPR protein linked to at least onenucleosome interacting protein domain, wherein the CRISPR protein (i)has nuclease or nickase activity or (ii) is modified to lack allnuclease activity and is linked to a non-nuclease domain; and (b) atleast one guide RNA or nucleic acid encoding at least one guide RNA;wherein the CRISPR protein of the at least one fusion protein istargeted to a target chromosomal sequence and the at least onenucleosome interacting protein domain of the at least one fusion proteinalters nucleosomal or chromatin structure such that the at least onefusion protein has increased access to the target chromosomal sequence,thereby increasing efficiency of targeted genome or epigeneticmodification.
 2. The method of claim 1, wherein the CRISPR protein is atype II CRISPR/Cas9 protein or a type V CRISPR/Cpf1 protein.
 3. Themethod of claim 1, wherein the non-nuclease domain has cytosinedeaminase activity, histone acetyltransferase activity, transcriptionalactivation activity, or transcriptional repressor activity.
 4. Themethod of claim 1, wherein the at least one nucleosome interactingprotein domain is a high mobility group (HMG) box (HMGB) DNA bindingdomain, a HMG nucleosome-binding (HMGN) protein, a central globulardomain from a histone H1 variant, a DNA binding domain from a chromatinremodeling complex protein, or a combination thereof.
 5. The method ofclaim 1, wherein the at least one nucleosome interacting protein domainis linked to the CRISPR protein directly via a chemical bond, indirectlyvia a linker, or a combination thereof.
 6. The method of claim 1,wherein the at least one nucleosome interacting protein domain is linkedto the CRISPR protein at its N-terminus, C-terminus, an internallocation, or a combination thereof.
 7. The method of claim 1, whereinthe at least one fusion protein further comprises at least one nuclearlocalization signal, at least one cell-penetrating domain, at least onemarker domain, or a combination thereof.
 8. The method of claim 1,wherein nucleic acid encoding the at least one fusion protein is codonoptimized for translation in the eukaryotic cell.
 9. The method of claim1, wherein nucleic acid encoding the at least one fusion protein is partof a viral vector, a plasmid vector, or a self-replicating RNA.
 10. Themethod of claim 1, wherein the method further comprises introducing intothe eukaryotic cell at least one donor polynucleotide, the donorpolynucleotide comprising at least one donor sequence.
 11. The method ofclaim 1, wherein the eukaryotic cell is in vitro.
 12. The method ofclaim 1, wherein the eukaryotic cell is in vivo.
 13. The method of claim1, wherein the eukaryotic cell is a mammalian cell.
 14. The method ofclaim 1, wherein the eukaryotic cell is a human cell.